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Sample records for activated recombinative desorption

  1. ACTIVATION ENERGY OF DESORPTION OF DIBENZOFURAN ON ACTIVATED CARBONS

    LI Xiang; LI Zhong; XI Hongxia; LUO Lingai

    2004-01-01

    Three kinds of commercial activated carbons, such as Norit RB1, Monolith and Chemviron activated carbons, were used as adsorbents for adsorption of dibenzofuran. The average pore size and specific surface area of these activated carbons were measured. Temperature Programmed Desorption (TPD) experiments were conducted to measure the TPD curves of dibenzofuran on the activated carbons, and then the activation energy for desorption of dibenzofuran on the activated carbons was estimated. The results showed that the Chemviron and the Norit RB1 activated carbon maintained higher specific surface area and larger micropore pore volume in comparison with the Monolith activated carbon, and the activation energy for the desorption of dibenzofuran on these two activated carbons was higher than that on the Monolith activated carbon. The smaller the pore of the activated carbon was, the higher the activated energy of dibenzofuran desorption was.

  2. Recombinant snake venom prothrombin activators

    Lövgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  3. Recombinant snake venom prothrombin activators.

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  4. ESTIMATION OF ACTIVATED ENERGY OF DESORPTION OF n-HEXANE ON ACTIVATED CARBONS BY TPD TECHNIQUE

    2001-01-01

    In this paper, six kinds of activated carbons such as Ag+-activated carbon, Cu2+-activated carbon, Fe3+- activated carbon, activated carbon, Ba2+- activated carbon and Ca2+-activated carbon were prepared. The model for estimating activated energy of desorption was established. Temperature-programmed desorption (TPD) experiments were conducted to measure the TPD curves of n-hexanol and then estimate the activation energy for desorption of n-hexanol on the activated carbons. Results showed that the activation energy for the desorption of n-hexanol on the Ag+- activated carbon, the Cu2+- activated carbon and the Fe3+- activated carbon were higher than those of n-hexanol on the activated carbon, the Ca2+- activated carbon and the Ba2+- activated carbon.

  5. ESTIMATION OF ACTIVATED ENERGY OF DESORPTION OF n—HEXANE ON ACTIVATED CARBONS BY PTD TECHNIQUE

    LIZhong; WANGHongjuan; 等

    2001-01-01

    In this paper,six kinds of activated carbons such as Ag+-activated carbon,Cu2+activated carbon,Fe3+-activated carbon,activated carbon,Ba2+-activated carbon and Ca2+activated carbon were prepared.The model for estimating activated energy of desorption was established.Temperature-programmed desorption(TPD)experiments were conducted to measure the TPD curves of n-hexanol and then estimate the activation energy for desorption of n-hexanol on the activated carbons.Results showed that the activation energy for the desorption of n-hexanol on the Ag+-activated carbon,the Cu2+-activated carbon and the Fe3+-activated carbon were higher than those of n-hexanol on the activated carbon,the Ca2+-activated carbon and the Ba2+-activated carbon.

  6. Magnetic monitoring of the hydrogenation-decomposition-desorption-recombination process in SmFe-based alloys

    A clear understanding of the reactions and phase changes that occur during the processing of a material is vital if we want to be able to achieve the best properties. In this series of experiments, we have used a vibrating-sample magnetometer (VSM) to monitor the magnetic state of compacted powder samples of Sm13.7Fe86.3 and Sm13.8Fe82.2Ta4.0 during a standard hydrogenation-disproportionation-desorption-recombination (HDDR) cycle. The samples were mounted in the VSM and heated at 4 deg.C/min in 1 bar of flowing hydrogen to 750 deg.C, they were then held for 60 min under hydrogen and approximately 120 min under vacuum before being cooled to room temperature under vacuum at 4 deg.C/min. The results show that the initial absorption of hydrogen by the Sm2Fe17-type phase results in an increase in magnetization as well as TC. At higher temperatures, the disproportionation reactions of the Sm13.7Fe86.3 and Sm13.8Fe82.2Ta4.0 materials were observed to proceed at a faster rate for the second and third cycles of the HDDR procedure than for the first. The results are in agreement with our previous transmission electron microscopy studies, which showed that much of the Ta that was initially dissolved in the Sm2Fe17-type phase of the cast Sm13.8Fe82.2Ta4.0 material was reformed as Ta-based precipitates after a single HDDR cycle leaving the Sm2Fe17-type phase with much less dissolved Ta

  7. Molecular requirements for radiation-activated recombination

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  8. Desorption of uranium from titanium-activated carbon composite adsorbent with acidic eluent, (1)

    An investigation was carried out on the desorption of uranium from titanium-activated carbon composite adsorbent with acidic eluent by the batch process. The rate of desorption of uranium with acidic eluent depended on temperature, showing an increase as the temperature was raised. But the rate of desorption with acidic eluent was less dependent on temperature than that obtained when mixed eluent of sodium carbonate-sodium hydrogencarbonate was used. The difference of the rate of desorption of uranium in the range of concentration from 0.3 to 0.5N was not found, and the rate of desorption with sulfuric acid was slightly higher than that obtained when hydrochloric acid was used. The amount of dissolved titanium decreased as the ratio of adsorbent to eluent (RAE) was increased. At RAE of 10%, the percentage of dissolved titanium (DTI) was below 0.38% with sulfuric acid, below 0.7% with hydrochloric acid. These values were found to be higher than the ones with the carbonate eluent. The elements except uranium, which were adsorbed on the adsorbent, were eluted simultaneously with acidic eluent. The regeneration of the adsorbent after desorption, therefore, was found to be unnecessary. In a repeated test of adsorption-desorption treatment up to five times, the percentage of uranium adsorbed from natural sea water was approximately constant of 85%. From these results, the application of column process to the desorption of uranium with acidic eluent at room temperature was proposed to be feasible. (author)

  9. Fibrinolytic Activity of Recombinant Mutant Streptokinase

    Mahboobeh Mobarrez

    2015-04-01

    Full Text Available Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity.

  10. Effect of soil type on adsorption-desorption, mobility, and activity of the herbicide norflurazon

    Morillo González, Esmeralda; Undabeytia López, Tomás; Cabrera Mesa, Alegría; Villaverde Capellán, J.; Maqueda Porras, Celia

    2004-01-01

    Adsorption-desorption studies of norflurazon on 17 soils of very different characteristics have been performed using a batch equilibration method and correlated to its mobility, activity, and persistence in soils. The influence of different soil properties and components on norflurazon adsorption was determined. The significant variables were organic matter (OM) content and iron and aluminum oxides, which accounted for 85 and 11% of the variability, respectively. Norflurazon desorption from s...

  11. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry

    Andersen, Jens S.; Svensson, B; Roepstorff, P

    1996-01-01

    Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy is...

  12. Effect of textural property of coconut shell-based activated carbon on desorption activation energy of benzothiophene

    Moxin YU; Zhong LI; Hongxia XI; Qibin XIA; Shuwen WANG

    2008-01-01

    In this work,the effect of the textural property of activated carbons on desorption activation energy and adsorption capacity for benzothiophene (BT) was investi-gated.BET surface areas and the textural parameters of three kinds of the activated carbons,namely SY-6,SY-13 and SY-19,were measured with an ASAP 2010 instru-ment.The desorption activation energies of BT on the activated carbons were determined by temperature-pro-grammed desorption (TPD).Static adsorption experi-ments were carried out to determine the isotherms of BT on the activated carbons.The influence of the textural property of the activated carbons on desorption activa-tion energy and the adsorption capacity for BT was dis-cussed.Results showed that the BET surface areas of the activated carbons,SY-6,SY-13 and SY-19 were 1106,diameters were 1.96,2.58 and 2.16 nm,respectively.The TPD results indicated that the desorption activation energy of BT on the activated carbons,SY-6,SY-19 and SY-13 were 58.84,53.02 and 42.57 KJ/mol,respectively.The isotherms showed that the amount of BT adsorbed on the activated carbons followed the order of SY-6 > SY-19 > SY-13.The smaller the average pore diameter of the activated carbon,the stronger its adsorption for BT and the higher the activation energy required for BT desorp-tion on its surface.The Freundlich adsorption isotherm model can be properly used to formulate the adsorption behavior of BT on the activated carbons.

  13. Impact of activated carbon, biochar and compost on the desorption and mineralization of phenanthrene in soil

    Marchal, Geoffrey; Smith, Kilian E.C.; Rein, Arno;

    2013-01-01

    Sorption of PAHs to carbonaceous soil amendments reduces their dissolved concentrations, limiting toxicity but also potentially biodegradation. Therefore, the maximum abiotic desorption of freshly sorbed phenanthrene (≤5 mg kg−1) was measured in three soils amended with activated carbon (AC...

  14. Helium Retention and Desorption Behaviour of Reduced Activation Ferritic/Martenstic Steel

    WANG Pinghuai; NOBUTA Yuji; HINO Tomoaki; YAMAUCHI Yuji; CHEN Zilning; XU Zengyu; LI Xiongwei; LIU Shi

    2009-01-01

    The reduced activation ferritic/martenstic steel CLF-1 prepared by the Southwest-ern Institute of Physics in China was irradiated by helium ions with an energy of 5 keV at room temperature using an electron cyclotron resonance (ECR) ion irradiation apparatus. After the irradiation, the helium retention and desorption were investigated using a technique of thermal desorption spectroscopy (TDS). The experiment was conducted with both the normal and welded samples. Blisters were observed after the helium ion irradiation, and the surface density of blisters in the welded samples was lower than that in the non-welded samples. Three desorption peaks were observed in both the non-welded and welded samples. These desorption peaks corresponded to those of blister ruptures and the helium release from the inner bubbles and the defects. The amount of helium retained in the welded samples was approximately the same as that in the non-welded samples, which was much less than other reduced activation materials, such as vanadium alloy and SiC/SiC composites.

  15. Desorption, partitioning, and dechlorination characteristics of PCBs in sediments in interaction with reactive activated carbon

    Choi, Hyeok, E-mail: hchoi@uta.edu [Department of Civil Engineering, The University of Texas at Arlington, 416 Yates Street, Arlington, TX 76019-0308 (United States); Environmental and Earth Sciences Program, The University of Texas at Arlington, 500 Yates Street, Arlington, TX 76019-0049 (United States); Lawal, Wasiu [Environmental and Earth Sciences Program, The University of Texas at Arlington, 500 Yates Street, Arlington, TX 76019-0049 (United States); Al-Abed, Souhail R. [National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 (United States)

    2015-04-28

    Highlights: • Problematic aged real PCBs-contaminated sediment (WHS) was examined. • Performance of reactive activated carbon (RAC) impregnated with Pd–ZVI was tested. • Fate and transport of PCBs bound to WHS in the presence of RAC was fully traced. • Direct mixing configuration was compared with compartment configuration. • Results reflected real world complexities associated with slow desorption of PCBs. - Abstract: Sediment (WHS) in Waukegan Harbor, Illinois, heavily contaminated and aged with polychlorinated biphenyls (PCBs), was treated with reactive activated carbon (RAC) impregnated with palladized iron nanoparticles. Lab test proceeded in a direct mixing configuration of RAC and WHS. A compartment configuration, where RAC was physically separated from WHS, was also designed to trace the sequential transport and fate of PCBs, including desorption, adsorption, dechlorination, and re-partitioning. PCBs, once desorbed from WHS, were immediately sequestrated to RAC and subject to dechlorination. Direct mixing of WHS with RAC was one-order of magnitude more effective for dechlorination than compartment configuration. Compared to their desorption-followed by-adsorption route, direct physical contact of RAC with PCBs bound to WHS exhibited negligible contribution to the availability of PCBs for dechlorination reaction. Addition of RAC even in compartment configuration facilitated PCBs desorption from WHS. However, slow desorption of PCBs limited overall performance, resulting in a five-order of magnitude lower dechlorination yield when compared with treatment of purely aqueous PCBs. The low dechlorination yield reflected real world complexities in treating 3.19% organic carbon-containing WHS aged with PCBs for 40 years. These observations were further supported when compared with results on clean Cesar Creek sediment spiked with 2-chlorinated biphenyls.

  16. Desorption, partitioning, and dechlorination characteristics of PCBs in sediments in interaction with reactive activated carbon

    Highlights: • Problematic aged real PCBs-contaminated sediment (WHS) was examined. • Performance of reactive activated carbon (RAC) impregnated with Pd–ZVI was tested. • Fate and transport of PCBs bound to WHS in the presence of RAC was fully traced. • Direct mixing configuration was compared with compartment configuration. • Results reflected real world complexities associated with slow desorption of PCBs. - Abstract: Sediment (WHS) in Waukegan Harbor, Illinois, heavily contaminated and aged with polychlorinated biphenyls (PCBs), was treated with reactive activated carbon (RAC) impregnated with palladized iron nanoparticles. Lab test proceeded in a direct mixing configuration of RAC and WHS. A compartment configuration, where RAC was physically separated from WHS, was also designed to trace the sequential transport and fate of PCBs, including desorption, adsorption, dechlorination, and re-partitioning. PCBs, once desorbed from WHS, were immediately sequestrated to RAC and subject to dechlorination. Direct mixing of WHS with RAC was one-order of magnitude more effective for dechlorination than compartment configuration. Compared to their desorption-followed by-adsorption route, direct physical contact of RAC with PCBs bound to WHS exhibited negligible contribution to the availability of PCBs for dechlorination reaction. Addition of RAC even in compartment configuration facilitated PCBs desorption from WHS. However, slow desorption of PCBs limited overall performance, resulting in a five-order of magnitude lower dechlorination yield when compared with treatment of purely aqueous PCBs. The low dechlorination yield reflected real world complexities in treating 3.19% organic carbon-containing WHS aged with PCBs for 40 years. These observations were further supported when compared with results on clean Cesar Creek sediment spiked with 2-chlorinated biphenyls

  17. Hydrogen desorption reactions of Li-N-H hydrogen storage system: Estimation of activation free energy

    The dehydrogenation reactions of the mixtures of lithium amide (LiNH2) and lithium hydride (LiH) were studied under an Ar atmosphere by means of temperature programmed desorption (TPD) technique. The dehydrogenation reaction of the LiNH2/LiH mixture was accelerated by addition of 1 mol% Ti(III) species (k = 3.1 x 10-4 s-1 at 493 K), and prolonged ball-milling time (16 h) further enhanced reaction rate (k = 1.1 x 10-3 s-1 at 493 K). For the hydrogen desorption reaction of Ti(III) doped samples, the activation energies estimated by Kissinger plot (95 kJ mol-1) and Arrhenius plot (110 kJ mol-1) were in reasonable agreement. The LiNH2/LiH mixture without Ti(III) species, exhibited slower hydrogen desorption process and the kinetic traces deviated from single exponential behavior. The results indicated the Ti(III) additives change the hydrogen desorption reaction mechanism of the LiNH2/LiH mixture

  18. Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures

    Andersen, Jens S.; Søgaard, M; Svensson, B;

    1994-01-01

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of peptide mixtures was used to characterize recombinant barley alpha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively, and...

  19. New aspects of Nd endash Fe endash B-based hydrogenation-disproportionation-desorption-recombination powders and anisotropic bonded magnets made from them: Microstructure and magnetic properties (invited)

    Recent progress in the understanding of the texture formation mechanism in the hydrogenation-disproportionation-desorption-recombination (HDDR) process, improvements of stability with regard to thermal and structural losses, and the state-of-the-art performance of both compression and injection molded magnets made from the newly developed anisotropic HDDR powders are reported. Transmission electron microscopy observations of disproportionated Nd endash Fe endash Co endash Ga endash Zr endash B alloys have revealed the existence of finely dispersed crystallites of Nd2(Fe,Co,Ga)14B which have a common crystallographic orientation. It is proposed that, upon removal of hydrogen, the hydrogen-disproportionated structure recombines from these crystallites to form textured submicron crystallites of the 2:14:1 phase. Using highly anisotropic HDDR powders, energy products (BH)max exceeding 170 kJ/m3 (21 MGOe) have been obtained on compression-molded resin-bonded magnets and 130 kJ/m3 (16 MGOe) on injection-molded ones. High coercivity HDDR powders with an intrinsic coercivity (HcJ) exceeding 1.27 MA/m (16 kOe) have also been obtained by replacing part of Nd with Dy, which enabled improvement of thermal stability. It is shown that the degradation of magnetic performance of resin-bonded HDDR magnets is prevented by eliminating pore formation during the molding process. copyright 1997 American Institute of Physics

  20. Biodegradation of persistent organics can overcome adsorption-desorption hysteresis in biological activated carbon systems.

    Abromaitis, V; Racys, V; van der Marel, P; Meulepas, R J W

    2016-04-01

    In Biological Activated Carbon (BAC) systems, persistent organic pollutants can be removed through a combination of adsorption, desorption and biodegradation. These processes might be affected by the presence of other organics, especially by the more abundant easily-biodegradable organics, like acetate. In this research these relations are quantified for the removal of the persistent pharmaceutical metoprolol. Acetate did not affect the adsorption and desorption of metoprolol, but it did greatly enhance the metoprolol biodegradation. At least part of the BAC biomass growing on acetate was also able to metabolise metoprolol, although metoprolol was only converted after the acetate was depleted. The presence of easily-degradable organics like acetate in the feeding water is therefore beneficial for the removal of metoprolol in BAC systems. The isotherms obtained from metoprolol adsorption and desorption experiments showed that BAC systems are subject to hysteresis; for AC bioregeneration to take place the microbial biomass has to reduce the concentration at the AC-biomass interface 2.7 times compared to the concentration at which the carbon was being loaded. However, given the threshold concentration of the MET degrading microorganisms (<0.08 μg/L) versus the average influent concentration (1.3 μg/L), bioregeneration is feasible. PMID:26855223

  1. Chi activity during transduction-associated recombination.

    Dower, N. A.; Stahl, F. W.

    1981-01-01

    Chi is a genetic element that stimulates phage lambda recombination by the Escherichia coli recBC pathway during lytic infection [Stahl, F. W. (1979) Annu. Rev. Genet. 13, 7--24]. Herein we show that chi in lambda prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation. This demonstration encourages the view that chi may influence genetic exchange in E. coli in the total absence of lambda.

  2. Diffusion barriers in the kinetics of water vapor adsorption/desorption on activated carbons

    Harding, A.W.; Foley, N.J.; Thomas, K.M. [Univ. of Newcastle upon Tyne (United Kingdom); Norman, P.R.; Francis, D.C. [CBD, Salisbury (United Kingdom)

    1998-07-07

    The adsorption of water vapor on a highly microporous coconut-shell-derived carbon and a mesoporous wood-derived carbon was studied. These carbons were chosen as they had markedly different porous structures. The adsorption and desorption characteristics of water vapor on the activated carbons were investigated over the relative pressure range p/p{degree} = 0--0.9 for temperatures in the range 285--313 K in a static water vapor system. The adsorption isotherms were analyzed using the Dubinin-Serpinski equation, and this provided an assessment of the polarity of the carbons. The kinetics of water vapor adsorption and desorption were studied with different amounts of preadsorbed water for set changes in pressure relative to the saturated vapor pressure (p/p{degree}). The adsorption kinetics for each relative pressure step were compared and used to calculate the activation energies for the vapor pressure increments. The kinetic results are discussed in relation to their relative position on the equilibrium isotherm and the adsorption mechanism of water vapor on activated carbons.

  3. Multiple biological activities of human recombinant interleukin 1.

    Dinarello, C A; Cannon, J. G.; Mier, J W; Bernheim, H. A.; LoPreste, G; Lynn, D L; Love, R N; Webb, A C; Auron, P. E.; Reuben, R C

    1986-01-01

    Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, wh...

  4. Adsorption and desorption of mixtures of organic vapors on beaded activated carbon.

    Wang, Haiyan; Jahandar Lashaki, Masoud; Fayaz, Mohammadreza; Hashisho, Zaher; Philips, John H; Anderson, James E; Nichols, Mark

    2012-08-01

    In this study, adsorption and desorption of mixtures of organic compounds commonly emitted from automotive painting operations were experimentally studied. A mixture of two alkanes and a mixture of eight organic compounds were adsorbed onto beaded activated carbon (BAC) and then thermally desorbed under nitrogen. Following both adsorption and regeneration, samples of the BAC were chemically extracted. Gas chromatography-mass spectrometry (GC-MS) was used to quantify the compounds in the adsorption and desorption gas streams and in the BAC extracts. In general, for both adsorbate mixtures, competitive adsorption resulted in displacing low boiling point compounds by high boiling point compounds during adsorption. In addition to boiling point, adsorbate structure and functionality affected adsorption dynamics. High boiling point compounds such as n-decane and 2,2-dimethylpropylbenzene were not completely desorbed after three hours regeneration at 288 °C indicating that these two compounds contributed to heel accumulation on the BAC. Additional compounds not present in the mixtures were detected in the extract of regenerated BAC possibly due to decomposition or other reactions during regeneration. Closure analysis based on breakthrough curves, solvent extraction of BAC and mass balance on the reactor provided consistent results of the amount of adsorbates on the BAC after adsorption and/or regeneration. PMID:22742925

  5. Thermal desorption of gases from aluminum alloy Al 6061, their rates and activation energies

    The use of Al in the construction of the vacuum chamber of intersecting proton storage rings has several advantages over the use of stainless steel. We have, therefore, undertaken UHV studies on long pipes made of Al alloy 6061 to see whether this material could be used in the proposed proton intersecting storage accelerator facility ISABELLE at Brookhaven. The outgassing rates, measured by two different methods, are compared for several surface treatments and three bakeout temperatures. The lowest outgassing rate of less than 1x10-14 Torr l s-1cm-2 was obtained with Ar and O2 discharge cleaning. The residual gas in the pipe contained more than 99% H2 with traces of CO and CO2. By observing the temperature dependence of the desorption of these gases, we have measured the heat of diffusion of H2 (E/sub H/2=10.5+-1.0 kcal/mole) and the thermal activation energies of CO (E/sub CO/=29+-2 kcal/mole) and CO2 (E/sub CO/2=25+-2 kcal/mole). Ar, with a peak desorption at 280degree--300 degreeC, also appears to be embedded in the metal. Since all commercially available UHV equipment uses stainless steel flanges, we have investigated the behavior of the stainless steel to Al transitions up to 350degreeC

  6. Visualization and Measurement of Adsorption/Desorption Process of Ethanol in Activated Carbon Adsorber

    Asano, Hitoshi; Murata, Kenta; Takenaka, Nobuyuki; Saito, Yasushi

    Adsorption refrigerator is one of the efficient tools for waste heat recovery, because the system is driven by heat at relative low temperature. However, the coefficient of performance is low due to its batch operation and the heat capacity of the adsorber. In order to improve the performance, it is important to optimize the configuration to minimize the amount of driving heat, and to clarify adsorption/desorption phenomena in transient conditions. Neutron radiography was applied to visualize and measure the adsorption amount distribution in an adsorber. The visualization experiments had been performed at the neutron radiography facility of E-2 port of Kyoto University Research Reactor. Activated carbon and ethanol were used as the adsorbent and refrigerant. From the acquired radiographs, adsorption amount was quantitatively measured by applying the umbra method using a checkered neutron absorber with boron powder. Then, transient adsorption and desorption processes of a rectangular adsorber with 84 mm in width, 50 mm in height and 20 mm in depth were visualized. As the result, the effect of fins in the adsorbent layer on the adsorption amount distribution was clearly visualized.

  7. A new method of regenerating activated carbon by thermal desorption with liquid water under subcritical conditions

    Salvador, F.; Sanchez Jimenez, C. [Universidad de Salamanca, Salamanca (Spain). Dept. de Quimica Fisica

    1996-06-01

    This paper discusses a new procedure for regenerating activated carbon based on thermal desorption with liquid water under subcritical conditions (300{degree}C and 120 atm). The method was assayed with three types of activated carbons exhausted with phenols (phenol and 4-nitrophenol), textile dyes (sirius red C.I.:29080 and orange II C.I.:15510) and pesticides (carbonfuran and atrazine). In all cases, total recovery of the adsorption capacity was achieved, even after seven regenerations. The mechanism and factors involved in regeneration were investigated using the TPD technique in liquid phase. The differences and advantages of this procedure as compared with others, such as thermal regeneration or regeneration with supercritical CO{sub 2} are discussed. 20 refs., 5 figs., 1 tab.

  8. Activity of recombinant factor VIIa under different conditions in vitro

    Bladbjerg, Else-Marie; Jespersen, Jørgen

    2008-01-01

    Recombinant activated factor VII (NovoSeven; Novo Nordisk A/S, Måløv, Denmark) is an effective drug for treatment of bleeding in patients with haemophilia A or B and inhibitors. Little is known about physiological conditions influencing the efficacy of recombinant activated factor VII. We...... investigated the in-vitro effects of pH, temperature, and haemodilution on the activity of recombinant activated factor VII. Samples from eight healthy volunteers were spiked with recombinant activated factor VII (final concentration 1.7 microg/ml) and adjusted to pH 6.0, 6.5, 7.0, and 7.4 or analysed at 30...... activity in plasma. Significant effects of pH were observed for clotting time, clot formation time, maximum clot firmness, and factor VII coagulant activity in the direction of longer clot formation times and less firm clots with decreasing pH. Temperature had significant effects on clotting time, clot...

  9. Modeling of hydrogen desorption from tungsten surface

    Hydrogen retention in metallic plasma-facing components is among key-issues for future fusion devices. For tungsten, which has been chosen as divertor material in ITER, hydrogen desorption parameters experimentally measured for fusion-related conditions show large discrepancies. In this paper, we therefore investigate hydrogen recombination and desorption on tungsten surfaces using molecular dynamics simulations and accelerated molecular dynamics simulations to analyze adsorption states, diffusion, hydrogen recombination into molecules, and clustering of hydrogen on tungsten surfaces. The quality of tungsten hydrogen interatomic potential is discussed in the light of MD simulations results, showing that three body interactions in current interatomic potential do not allow to reproduce hydrogen molecular recombination and desorption. Effects of surface hydrogen clustering on hydrogen desorption are analyzed by introducing a kinetic model describing the competition between surface diffusion, clustering and recombination. Different desorption regimes are identified and reproduce some aspects of desorption regimes experimentally observed

  10. Modeling of hydrogen desorption from tungsten surface

    Guterl, J., E-mail: jguterl@ucsd.edu [University of California, San Diego, La Jolla, CA 92093 (United States); Smirnov, R.D. [University of California, San Diego, La Jolla, CA 92093 (United States); Krasheninnikov, S.I. [University of California, San Diego, La Jolla, CA 92093 (United States); Nuclear Research National University MEPhI, Moscow 115409 (Russian Federation); Uberuaga, B.; Voter, A.F.; Perez, D. [Los Alamos National Laboratory, Los Alamos, NM 8754 (United States)

    2015-08-15

    Hydrogen retention in metallic plasma-facing components is among key-issues for future fusion devices. For tungsten, which has been chosen as divertor material in ITER, hydrogen desorption parameters experimentally measured for fusion-related conditions show large discrepancies. In this paper, we therefore investigate hydrogen recombination and desorption on tungsten surfaces using molecular dynamics simulations and accelerated molecular dynamics simulations to analyze adsorption states, diffusion, hydrogen recombination into molecules, and clustering of hydrogen on tungsten surfaces. The quality of tungsten hydrogen interatomic potential is discussed in the light of MD simulations results, showing that three body interactions in current interatomic potential do not allow to reproduce hydrogen molecular recombination and desorption. Effects of surface hydrogen clustering on hydrogen desorption are analyzed by introducing a kinetic model describing the competition between surface diffusion, clustering and recombination. Different desorption regimes are identified and reproduce some aspects of desorption regimes experimentally observed.

  11. Production of biologically active recombinant human lactoferrin in Aspergillus oryzae.

    Ward, P P; Lo, J Y; Duke, M; May, G S; Headon, D R; Conneely, O M

    1992-07-01

    We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae alpha-amylase promoter and the 3' flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity. The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins. PMID:1368268

  12. 4-MCHM sorption to and desorption from granular activated carbon and raw coal.

    Jeter, T Scott; Sarver, Emily A; McNair, Harold M; Rezaee, Mohammad

    2016-08-01

    4-Methylcyclohexanemethanol (4-MCHM) is a saturated higher alicyclic primary alcohol that is used in the froth flotation process for cleaning coal. In early 2014, a large spill of crude chemical (containing primarily 4-MCHM) to the Elk River near Charleston, WV contaminated the local water supply. Carbon filters at the affected water treatment facility quickly became saturated, and the contaminated water was distributed to nearby homes and businesses. Sorption of 4-MCHM to granular activated carbon (GAC) was studied in the laboratory using head space (HS) analysis via gas chromatography with a flame ionization detector (GC-FID). Sorption to raw coal was also investigated, since this material may be of interest as a sorbent in the case of an on-site spill. As expected, sorption to both materials increased with decreased particle size and with increased exposure time; although exposure time proved to be much more important in the case of GAC than for coal. Under similar conditions, GAC sorbed more 4-MCHM than raw coal (e.g., 84.9 vs. 63.1 mg/g, respectively, for 20 × 30 mesh particles exposed to 860 mg/L 4-MCHM solution for 24 h). Desorption from both materials was additionally evaluated. Interestingly, desorption of 4-MCHM on a mass per mass basis was also higher for GAC than for raw coal. Overall, results indicated that GAC readily sorbs 4-MCHM but can also readily release a portion of the chemical, whereas coal sorbs somewhat less 4-MCHM but holds it tightly. PMID:27219291

  13. Desorption experiments and modeling of micropollutants on activated carbon in water phase: application to transient concentrations mitigation

    Bourneuf, Séda; Jacob, Matthieu; Albasi, Claire; Sochard, Sabine; Richard, Romain; Manero, Marie-Hélène

    2016-01-01

    International audience Experimental studies and numerical modeling were conducted to assess the feasibility of a granular activated carbon column to buffer load variations of contaminants before wastewater treatment devices. Studies of cycles of adsorption, and more especially desorption, of methyldiethanolamine (MDEA) and 2,4-dimethylphenol (2,4-DMP) have been carried out on granular activated carbon (GAC). Dynamic variations of contaminants concentrations were run at several conditions o...

  14. Deuterium thermal desorption from FeAl thin films

    Deuterium thermal desorption experiments were performed on B2 FeAl thin films deposited by electron-beam evaporation on Si substrates, annealed at temperatures ranging from 673 to 773 K and implanted with 20 keV D2 ions at fluences of 3x1016 D2+ cm-2. D2 desorption spectra, recorded in isochronal heating conditions (0.5 K s-1 temperature ramp), show two well resolved desorption peaks at ∼500 and 820 K. The first peak is attributed to the desorption of deuterium contained in FeAl weakly bonding sites of the FeAl lattice, probably point defects (vacancies) produced by the ion implantation process. The second peak is attributed to deuterium release from trapping sites in the FeAl lattice that could be the defect complexes formed by the association of a vacancy in the Fe sublattice and a substitutional Fe atom in the Al sublattice (VFe-FeAl). The desorption kinetics can be reproduced by assuming that: (a) deuterium desorption at ∼500 K is controlled by the D2 surface recombination process with Edes=1.57±0.02 eV as activation energy; (b) deuterium desorption at ∼820 K is controlled by the release of D atoms from the trap sites, a process which occurs in connection with defect relaxation. The energy of interaction of deuterium with this trapping site can be estimated to be ∼2 eV. (author)

  15. Theoretical and Experimental Study on the Adsorption and Desorption of Methane by Granular Activated Carbon at 25 ℃

    E. Salehi; V. Taghikhani; C. Ghotbi; E. Nemati Lay; A. Shojaei

    2007-01-01

    A theoretical and experimental study was conducted to accurately determine the amount of adsorption and desorption of methane by various Granular Activated Carbon (GAC) under different physical conditions. To carry out the experiments, the volumetric method was used up to 500 psia at constant temperature of 25 ℃. In these experiments, adsorption as well as desorption capacities of four different GAC in the adsorption of methane, the major constituent of natural gas, at various equilibrium pressures and a constant temperature were studied. Also, various adsorption isotherm models were used to model the experimental data collected from the experiments. The accuracy of the results obtained from the adsorption isotherm models was compared and the values for the regressed parameters were reported. The results shows that the physical characteristics of activated carbons such as BET surface area, micropore volume, packing density, and pore size distribution play an important role in the amount of methane to be adsorbed and desorbed.

  16. Recombinant tissue plasminogen activator following paediatric cataract surgery

    Mehta, J; ADAMS, G

    2000-01-01

    BACKGROUND—The use of recombinant tissue plasminogen activator (r-TPA) has been advocated in the treatment of postsurgical fibrinous membrane formation following cataract surgery in adults. Its use in paediatric cases is not well documented.
METHOD—A retrospective review of paediatric cataract extractions performed at Moorfields Eye Hospital between 1 January 1997 and 4 April 1999 was carried out.
RESULTS—Cataract extractions were performed in 37 patients, 22 in males 15 in females. Four (9.2...

  17. Effects and Mechanisms of Mechanical Activation on Hydrogen Sorption/ Desorption of Nanoscale Lithium Nitrides

    Shaw, Leon, L.; Yang, Gary, Z.; Crosby, Kyle; Wwan, Xufei. Zhong, Yang; Markmaitree, Tippawan; Osborn, William; Hu, Jianzhi; Kwak, Ja Hun

    2012-04-26

    The objective of this project is to investigate and develop novel, mechanically activated, nanoscale Li3N-based and LiBH4-based materials that are able to store and release {approx}10 wt% hydrogen at temperatures near 100 C with a plateau hydrogen pressure of less than 10 bar. Four (4) material systems have been investigated in the course of this project in order to achieve the project objective. These 4 systems are (i) LiNH2+LiH, (ii) LiNH2+MgH2, (iii) LiBH4, and (iv) LiBH4+MgH2. The key findings we have obtained from these 4 systems are summarized below. *The thermodynamic driving forces for LiNH2+LiH and LiBH4 systems are not adequate to enable H2 release at temperatures < 100 C. *Hydrogen release in the solid state for all of the four systems is controlled by diffusion, and thus is a slow process. *LiNH2+MgH2 and LiBH4+MgH2 systems, although possessing proper thermodynamic driving forces to allow for H2 release at temperatures < 100 C, have sluggish reaction kinetics because of their diffusion-controlled rate-limiting steps. *Reducing particles to the nanometer length scale (< 50 nm) can improve the thermodynamic driving force to enable H2 release at near ambient temperature, while simultaneously enhancing the reaction kinetics as well as changing the diffusion-controlled rate-limiting step to gas desorption-controlled rate-limiting step. This phenomenon has been demonstrated with LiBH4 and offers the hope that further work along this direction will make one of the material systems, i.e., LiBH4, LiBH4+MgH2 and LiNH2+MgH2, possess the desired thermodynamic properties and rapid H2 uptake/release kinetics for on-board applications. Many of the findings and knowledge gained from this project have been published in archival refereed journal articles [1-15] and are accessible by general public. Thus, to avoid a bulky final report, the key findings and knowledge gained from this project will be succinctly summarized, particularly for those findings and knowledge

  18. Three faces of recombination activating gene 1 (RAG1) mutations.

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation. PMID:26689875

  19. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  20. Improving of understanding of beta-hexachlorocyclohexane (HCH) adsorption on activated carbons by temperature-programmed desorption studies.

    Passé-Coutrin, Nady; Maisonneuve, Laetitia; Durimel, Axelle; Dentzer, Joseph; Gadiou, Roger; Gaspard, Sarra

    2016-01-01

    In order to understand the interactions between beta-hexachlorocyclohexane (HCH) and chemical groups at activated carbon (AC) surface, the solid samples were hydrogenated aiming to decrease the amounts of oxygenated groups. Two AC samples designated by BagH2O and BagP1.5 were prepared by water vapor activation and phosphoric acid activation, respectively, of sugarcane bagasse used as an AC precursor. A more simple molecule 1,2,3-trichloropropane (TCP) is used as a model of chlorinated compound. The AC were characterized by infrared, X-ray photoelectron spectroscopy (XPS), Raman resonance spectroscopies, as well as temperature-programmed desorption coupled with mass spectrometry (TPD-MS). BagP1.5 and BagH2O AC surface contained oxygenated groups. Upon hydrogenation, a decrease of most of these group amxounts was observed for both samples, while hydroxyl groups increased. On the basis of temperature-programmed desorption data obtained for AC samples contaminated with TCP or HCH, it was possible to determine the type of hydrogen bond formed between each AC and HCH. PMID:26018287

  1. 78 FR 27977 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Actions Under the NIH...

    2013-05-13

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... Acid Molecules (NIH Guidelines) SUMMARY: The NIH Office of Biotechnology Activities (NIH OBA) proposes... by mail to the NIH Office of Biotechnology Activities, National Institutes of Health, 6705...

  2. 75 FR 28811 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Actions Under the NIH...

    2010-05-24

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... Office of Biotechnology Activities (OBA) by the Institutional Biosafety Committee at Lawrence Livermore... Biotechnology Activities, National Institutes of Health. BILLING CODE 4140-01-P...

  3. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    2011-07-25

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... Minor Action under the NIH Guidelines. SUMMARY: The Office of Biotechnology Activities (OBA) is updating... Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, Bethesda,...

  4. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    Johansson, Pär I; Ostrowski, Sisse R

    2010-01-01

    Recombinant activated factor VII (rFVIIa, NovoSeven) was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.......Recombinant activated factor VII (rFVIIa, NovoSeven) was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX....

  5. Recombinant human migration inhibitory factor has adjuvant activity.

    Weiser, W Y; Pozzi, L M; Titus, R G; David, J R

    1992-01-01

    Recombinant human migration inhibitory factor (MIF), isolated through functional expression cloning in COS-1 cells, up-regulates expression of genes encoding HLA-DR and interleukin 1 beta (IL-1 beta) and elaboration of IL-1 beta by human monocyte-derived macrophages. Administration of soluble bovine serum albumin or human immunodeficiency virus 120-kDa glycoprotein (HIV gp120) to mice in the presence of recombinant MIF together with incomplete Freund's adjuvant induced a strong T-cell prolife...

  6. Myelostimulatory activity of recombinant human interleukin-2 in mice

    In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy

  7. Deuterium thermal desorption from FeAl thin films

    Checchetto, R. [Istituto Nazionale per la Fisica della Materia (INFM), Dipartimento di Fisica dell' Universita di Trento, Povo (Italy)]. E-mail: checchet@science.unitn.it; Miotello, A.; Tosello, C. [Istituto Nazionale per la Fisica della Materia (INFM), Dipartimento di Fisica dell' Universita di Trento, Povo (Italy); Principi, G. [Istituto Nazionale per la Fisica della Materia (INFM), Dipartimento di Ingegneria Meccanica e Strutturale dell' Universita di Padova, Padova (Italy); Mengucci, P. [Istituto Nazionale per la Fisica della Materia (INFM), Dipartimento di Fisica e Ingegneria dei Materiali e del Territorio dell' Universita di Ancona, Ancona (Italy)

    2002-07-01

    Deuterium thermal desorption experiments were performed on B2 FeAl thin films deposited by electron-beam evaporation on Si substrates, annealed at temperatures ranging from 673 to 773 K and implanted with 20 keV D{sub 2} ions at fluences of 3x10{sup 16} D{sub 2}{sup +} cm{sup -2}. D{sub 2} desorption spectra, recorded in isochronal heating conditions (0.5 K s{sup -1} temperature ramp), show two well resolved desorption peaks at {approx}500 and 820 K. The first peak is attributed to the desorption of deuterium contained in FeAl weakly bonding sites of the FeAl lattice, probably point defects (vacancies) produced by the ion implantation process. The second peak is attributed to deuterium release from trapping sites in the FeAl lattice that could be the defect complexes formed by the association of a vacancy in the Fe sublattice and a substitutional Fe atom in the Al sublattice (V{sub Fe}-Fe{sub Al}). The desorption kinetics can be reproduced by assuming that: (a) deuterium desorption at {approx}500 K is controlled by the D{sub 2} surface recombination process with E{sub des}=1.57{+-}0.02 eV as activation energy; (b) deuterium desorption at {approx}820 K is controlled by the release of D atoms from the trap sites, a process which occurs in connection with defect relaxation. The energy of interaction of deuterium with this trapping site can be estimated to be {approx}2 eV. (author)

  8. Supercritical fluid extraction and temperature-programmed desorption of phenol and its oxidative coupling products from activated carbon

    Humayun, R.; Karakas, G.; Dahlstrom, P.R.; Ozkan, U.S.; Tomasko, D.L. [Ohio State Univ., Columbus, OH (United States). Dept. of Chemical Engineering

    1998-08-01

    Activated carbon remains one of the most economical adsorbents for the removal of contaminants from water. In particular, activated carbon is known to have an extremely high affinity for phenol and its derivatives. This has been shown to be the result of a catalytic process wherein activated carbon catalyzes the oxidative coupling reactions of phenol in aqueous solution when molecular oxygen is present. These reactions are believed to be the source of the difficulty of regenerating activated carbon loaded with phenol. This paper reports on efforts toward using supercritical fluids to regenerate activated carbon combined with a concurrent temperature-programmed desorption study to identify reaction products and their binding strength to the carbon surface. The results show unequivocally that part of the phenol is chemisorbed on the surface and part of it undergoes polymerization. Dihydroxybiphenyls and phenoxyphenols are the major reaction products present on the surface. Isotope studies showed that surface carbon atoms do not directly participate in these reactions. Supercritical extraction was found to perform as well as solvent extraction for the regeneration of activated carbon loaded with phenol. However, due to the chemisorbed nature of these oxidative coupling products, the reduced mass-transfer limitations afforded by supercritical extraction cannot improve the overall extent of extraction even though the rate is improved with the addition of cosolvents.

  9. Biodegradation of high explosives on granular activated carbon [GAC]: Enhanced desorption of high explosives from GAC -- Batch studies

    Morley, M.C.; Speitel, G.E. Jr. [Univ. of Texas, Austin, TX (United States). Dept. of Civil Engineering

    1999-03-01

    Adsorption to GAC is an effective method for removing high explosives (HE) compounds from water, but no permanent treatment is achieved. Bioregeneration, which treats adsorbed contaminants by desorption and biodegradation, is being developed as a method for reducing GAC usage rates and permanently degrading RDX and HMX. Because desorption is often the limiting mass transfer mechanism in bioregeneration systems, several methods for increasing the rate and extent of desorption of RDX and HMX are being studied. These include use of cosolvents (methanol and ethanol), surfactants (both anionic and nonionic), and {beta}- and {gamma}-cyclodextrins. Batch experiments to characterize the desorption of these HEs from GAC have been completed using Northwestern LB-830, the GAC being used at Pantex. Over a total of 11 days of desorption, about 3% of the adsorbed RDX was desorbed from the GAC using buffered water as the desorption fluid. In comparison, about 96% of the RDX was extracted from the GAC by acetonitrile over the same desorption period. Ethanol and methanol were both effective in desorbing RDX and HMX; higher alcohol concentrations were able to desorb more HE from the GAC. Surfactants varied widely in their abilities to enhance desorption of HEs. The most effective surfactant that was studied was sodium dodecyl sulfate (SDS), which desorbed 56.4% of the adsorbed RDX at a concentration of 500 mg SDS/L. The cyclodextrins that were used were marginally more effective than water. Continuous-flow column tests are underway for further testing the most promising of these methods. These results will be compared to column experiments that have been completed under baseline conditions (using buffered water as the desorption fluid). Results of this research will support modeling and design of further desorption and bioregeneration experiments.

  10. Use of thermal desorption gas chromatography-olfactometry/mass spectrometry for the comparison of identified and unidentified odor active compounds emitted from building products containing linseed oil

    Clausen, P. A.; Knudsen, Henrik Nellemose; Larsen, K.;

    2008-01-01

    The emission of odor active volatile organic compounds (VOCs) from a floor oil based on linseed oil, the linseed oil itself and a low-odor linseed oil was investigated by thermal desorption gas chromatography combined with olfactometry and mass spectrometry (TD-GC-O/MS). The oils were applied to...

  11. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

    Sun, Bo; Li, Zhao-Shen; Tu, Zhen-Xing; Xu, Guo-Ming; Du, Yi-qi

    2006-01-01

    AIM: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity.

  12. An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

    Marcio Jose Poças-Fonseca; Ildinete Silva-Pereira; Cynthia Maria Kyaw; Edivaldo Ximenes Ferreira Filho; Fabrícia Paula de Faria; Gilvan Caetano Duarte; Marciano Regis Rubini; Thiago Machado Mello-de-Sousa; Eveline Queiroz de Pinho Tavares

    2013-01-01

    Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was dete...

  13. Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

    Yan, Jing; Gong, Yuewen; She, Yi-Min; Wang, Guqi; Roberts, Michael S; Burczynski, Frank J.

    2009-01-01

    Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrup...

  14. Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi

    Geldhof, Peter; Meyvis, Yves; Vercruysse, Jozef; Claerebout, Edwin

    2008-01-01

    Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challeng...

  15. The negligible photodesorption of methanol ice and the active photon-induced desorption of its irradiation products

    Cruz-Diaz, Gustavo A; Caro, Guillermo M Muñoz; Chen, Y -J

    2016-01-01

    Methanol is a common component of interstellar and circumstellar ice mantles and is often used as an evolution indicator in star-forming regions. The observations of gas-phase methanol in the interiors of dense molecular clouds at temperatures as low as 10 K suggests that a non-thermal ice desorption must be active. Ice photodesorption was proposed to explain the abundances of gas-phase molecules toward the coldest regions. Laboratory experiments were performed to investigate the potential photodesorption of methanol toward the coldest regions. Solid methanol was deposited at 8 K and UV-irradiated at various temperatures starting from 8 K. The irradiation of the ice was monitored by means of infrared spectroscopy and the molecules in the gas phase were detected using quadrupole mass spectroscopy. Fully deuterated methanol was used for confirmation of the results. The photodesorption of methanol to the gas phase was not observed in the mass spectra at different irradiation temperatures. We estimate an upper li...

  16. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    2010-11-15

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities Recombinant DNA Research... Biotechnology Activities (OBA). The data to be considered for certifying a new host-vector system can be found... 301-496-9839 or sent by U.S. mail to the Office of Biotechnology Activities, National Institutes...

  17. Activated desorption at heterogeneous interfaces and long-time kinetics of hydrocarbon recovery from nanoporous media

    Lee, Thomas; Bocquet, Lydéric; Coasne, Benoit

    2016-06-01

    Hydrocarbon recovery from unconventional reservoirs (shale gas) is debated due to its environmental impact and uncertainties on its predictability. But a lack of scientific knowledge impedes the proposal of reliable alternatives. The requirement of hydrofracking, fast recovery decay and ultra-low permeability--inherent to their nanoporosity--are specificities of these reservoirs, which challenge existing frameworks. Here we use molecular simulation and statistical models to show that recovery is hampered by interfacial effects at the wet kerogen surface. Recovery is shown to be thermally activated with an energy barrier modelled from the interface wetting properties. We build a statistical model of the recovery kinetics with a two-regime decline that is consistent with published data: a short time decay, consistent with Darcy description, followed by a fast algebraic decay resulting from increasingly unreachable energy barriers. Replacing water by CO2 or propane eliminates the barriers, therefore raising hopes for clean/efficient recovery.

  18. Transcriptional activities of mammalian genomes at sites of recombination with foreign DNA

    The nucleotide sequences of several sites of recombination between adenovirus DNA and hamster, mouse, or human cell DNAs were determined. These sites of recombination had been cloned from adenovirus type 2 (Ad2)- or type 12 (Ad12)-transformed cells, from Ad12-induced tumor cells, or from a symmetric recombinant between Ad12 DNA and human cell DNA. One important precondition for the generation of recombinants between host and foreign DNAs might be the establishment of a chromatin configuration that permits access of foreign DNA and of the recombination machinery to cellular DNA. Such favorable chromatin structures might arise during cellular DNA replication or transcription or both. As a first approach toward investigating these more complex problems of foreign DNA insertion. The authors determined transcriptional activities of cellular DNA sequences at viral junction sites. The results presented demonstrate that the cellular DNA sequences involved in linkage to viral DNA at five completely different sites in DNA from three different species are transcribed into RNAs even in cells which have not been transformed or infected by adenovirus. These results are consistent with the notion that at least some of the cellular DNA sequences at sites of insertion of adenovirus (foreign) DNA are transcriptionally active and thus provide an opportunity for recombination

  19. Purification and Determination of Inhibitory Activity of Recombinant Soyacystatin Against Papain and Fish Protease

    AKPINAR, Özlem

    2004-01-01

    Recombinant (r-) soyacystatin was characterized for its inhibitory activity against papain and compared to egg white cystatin. r-Soyacystatin expressed in E. coli was purified with phenyl-Sepharose and DEAE 4.33 fold as a recombinant protein. Egg white cystatin was purified by using affinity chromatography on cm-papain-Sepharose. Inhibitory activity of r-soyacystatin was similar to that of egg white cystatin. The amount required to inhibit 50% activity of papain used in the assay, 2 pg, was 0...

  20. Synergistic hydrogen desorption behavior of magnesium aluminum hydride synthesized by mechano-chemical activation method

    Highlights: ► Mg(AlH4)2 could release 3.1 wt% H2 at initial temperature as low as 100 °C. ► The synergistic and mutual catalytic effects of NaAlH4 and Mg(AlH4)2 were noticed. ► In situ synchrotron XRD confirmed the two-step dehydrogenation process of Mg(AlH4)2. - Abstract: A mechano-chemical activation synthesis (MCAS) is employed to fabricate Mg(AlH4)2 via milling the precursors, specifically NaAlH4 and MgCl2. The corresponding dehydrogenation behavior of the synthesized powders is investigated. The experimental results showed that incomplete synthesis or premature dehydrogenation may occur if the milling process was not properly controlled. The hydrogen content of each synthesized powder is determined by using a thermal gravimetric analyzer (TGA). The dehydrogenation reactions of the synthesized powders are investigated by employing ex situ X-ray diffraction (XRD), in situ synchrotron XRD and differential thermal analysis (DTA). The results showed that the incompletely synthesized powder consisted of residual NaAlH4 in the synthesized Mg(AlH4)2, which demonstrated an initial dehydrogenation temperature as low as 100 °C and accompanied with a maximum amount (3.1 wt%) of H2 released below 350 °C. The mutual catalytic effect of both NaAlH4 and Mg(AlH4)2 on lowering their initial dehydrogenation temperature is confirmed.

  1. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    2010-07-20

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... transgenic rodent and a non-transgenic rodent). The NIH Office of Biotechnology Activities (OBA) received a... to the same email address or by fax to 301-496-9839 or mail to the Office of Biotechnology...

  2. 78 FR 12074 - Office of Biotechnology Activities; Recombinant DNA Research: Actions Under the NIH Guidelines...

    2013-02-21

    ... containing an HA from the Goose/Guangdong/1/96 lineage should become an HHS Select Agent (77 FR 63783... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... recommendations of the RAC, the NIH Office of Biotechnology Activities (OBA) concluded that more specific...

  3. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    2011-01-19

    ...). On July 20, 2010 the NIH Office of Biotechnology Activities (OBA) published a proposed action (75 FR... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... contact OBA by e- mail at oba@od.nih.gov , telephone, 301-496-9838 or mail to the Office of...

  4. 76 FR 27653 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    2011-05-12

    ... Federal Register (75 FR 69687). No public comments were received regarding the proposal to certify K... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA.... lactis certified host-vector 1 system. In addition, the Office of Biotechnology Activities is...

  5. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    2010-06-04

    ..., 2010 (75 FR 28811) is withdrawn. The discussion that was to be held at the June 16-17, 2010 meeting of... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA...-Curay, Acting Director, Office of Biotechnology Activities, National Institutes of Health. BILLING...

  6. 76 FR 62816 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    2011-10-11

    ... experts from NIH, CDC, and academia. These proposed changes were published in the Federal Register (76 FR... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... Biotechnology Activities (OBA) is updating Appendix B of the NIH Guidelines to specify the risk group...

  7. Antibacterial Activity of Recombinant Pig Intestinal Parasite Cecropin P4 Peptide Secreted from Pichia pastoris.

    Song, Ki-Duk; Lee, Woon-Kyu

    2014-02-01

    Cecropins (Cec) are antibacterial peptides and their expression is induced in a pig intestinal parasite Ascaris suum by bacterial infection. To explore the usefulness of its activity as an antibiotic, CecP4 cDNA was prepared and cloned into the pPICZ B expression vector and followed by the integration into AOX1 locus in Pichia pastoris. The supernatants from cell culture were collected after methanol induction and concentrated for the test of antimicrobial activity. The recombinant P. patoris having CecP4 showed antimicrobial activity when tested against Staphyllococcus aureus in disc diffusion assay. We selected one of the CecP4 clones (CecP4-2) and performed further studies with it. The growth of recombinant P. pastoris was optimized using various concentration of methanol, and it was found that 2% methanol in the culture induced more antibacterial activity, compared to 1% methanol. We extended the test of antimicrobial activity by applying the concentrated supernatant of CecP4 culture to Pseudomonas aeruginosa and E. coli respectively. Recombinant CecP4 also showed antimicrobial activity against both Pseudomona and E. coli, suggesting the broad spectrum of its antimicrobial activity. After improvements for the scale-up, it will be feasible to use recombinant CecP4 for supplementation to the feed to control microbial infections in young animals, such as piglets. PMID:25049952

  8. Synthesis and characterization of biologically active recombinant elk and horse FSH.

    Fachal, María Victoria; Furlan, Mike; Clark, Rena; Card, Claire E; Chedrese, P Jorge

    2010-02-01

    The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well. PMID:19500922

  9. Experimental and Theoretical Study of the Effect of Moisture on Methane Adsorption and Desorption by Activated Carbon at 273.5 K

    2007-01-01

    Adsorption and desorption of methane by activated carbon (AC) at constant temperature and at various pressures were investigated. The effect of moisture was also studied. A volumetric method was used, up to 40 bar, at a temperature of 273.5 K. Results of a dry AC sample were compared with those obtained from a moist sample and two different ACs with different physical and surface properties were used. As expected, the results showed that the existence of moisture, trapped in the AC pores, could lead to a decrease in the amount of methane adsorbed and a decrease in the amount of methane delivered during desorption. To model the experimental results, a large variety of adsorption isotherms were used. The regressed parameters for the adsorption isotherms were obtained using the experimental data generated in the present study. The accuracy of the results obtained from the different adsorption isotherms was favorably compared.

  10. Sorption-Desorption Behavior of Newly synthesized N-(1H-Benzimidazole -2 ylmethyl) Acetamide (ABNZ) on Selected Soils and its Antifungal activity

    A batch equilibrium method has been utilized to investigate the adsorption-desorption behavior of a versatile commercially available fungicide N-(1H-benzimidazole-2ylmethyl) acetamide (ABNZ) on four Pakistani soils geographically distant, from hilly to desert areas. Both qualitative and quantitative analysis has been done for sorption-desorption behavior of ABNZ. These analyses are done in the different concentration of fungicide (0-0.25, 0.25-0.5, 0.5-0.75). Based on statistical analysis ABNZ execute the Freundlich adsorption isotherm in all four tested soils having values of slope n<1 resembling the L-type curve. The distribution co-efficient K/sub d(ads)/ ranges from (2.66 to 12.42 ml/meu g/sup -1/) indicating low adsorption of ABNZ overall. Sorption increases with soil organic carbon content, exhibited greater degree of adsorption for hilly soil and least adsorption on sandy soil of Multan, Punjab. Desorption studies reveal that the adsorbed fungicide is firmly retained by soil particles and present a certain degree of irreversibility. The results indicate that the soil organic matters followed by clay content are the most important soil properties governing the fungicide sorption capacity. Compound N-(1H- benzimidazole -2-ylmethyl) acetamide (ABNZ) showed maximum antifungal activity compared to rest of the tested compound. This anti-fungal activity is substantial in comparison to other pesticides. It causes 40% inhibition of Microsporum canis and Fusarium solani. (author)

  11. Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish

    Proaerolysin-coding gene was cloned from the genomic DNA of A. hydrophila and heterologously expressed in E. coli. The purified recombinant proaerolysin was inactive and could be activated by treatment with proteases, furin and trypsin, and extra-cellular proteins (ECPs, the cell-free supernatant of...

  12. Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus.

    Johnson, T M; Mann, W R; Dragland, C J; Anderson, R C; Nemecek, G M; Bell, P A

    1995-01-01

    The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively. Images Figure 1 Figure 2 Figure 4 PMID:7626037

  13. Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

    M. Schouten; C. van 't Veer; J.J.T.H. Roelofs; B. Gerlitz; B.W. Grinnell; M. Levi; T. van der Poll

    2011-01-01

    Recombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associate

  14. [Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

    Fan, Fangfang; Sun, Huiying; Xu, Hui; Liu, Jiawei; Zhang, Haiyuan; Li, Yilan; Ning, Xuelian; Sun, Yue; Bai, Jing; Fu, Songbin; Zhou, Chunshui

    2015-12-01

    AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future. PMID:27093838

  15. Effect of copper on the recombination activity of extended defects in silicon

    The effect of copper atoms introduced by high-temperature diffusion on the recombination properties of dislocations and dislocation trails in p-type single-crystal silicon is studied by the electron-beam-induced current technique. It is shown that, in contrast to dislocations, dislocation trails exhibit an increase in recombination activity after the introduction of copper. Bright contrast appearance in the vicinity of dislocation trails is detected after the diffusion of copper and quenching of the samples. The contrast depends on the defect density in these trails

  16. Role of recombination activating genes in the generation of antigen receptor diversity and beyond.

    Nishana, Mayilaadumveettil; Raghavan, Sathees C

    2012-12-01

    V(D)J recombination is the process by which antibody and T-cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non-homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence-specific nuclease and its pathological role as a structure-specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence-specific and structure-specific nuclease. It also deals with the off-target cleavage of RAGs and its implications in genomic instability. PMID:23039142

  17. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity.

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

  18. Antitumor activity of recombinant interleukin 6 in mice

    1990-01-01

    IL-6 possesses multiple biologic activities that affect a broad range of cells including those directly involved in immune responses as well as cells important in the systemic response to infection or trauma. We now show that purified human rIL-6, when administered alone at relatively high doses that are comparable to therapeutic levels of IL- 2, mediated substantial reductions in the number of pulmonary and hepatic micrometastases from four distinct syngeneic tumors. Unlike IL- 2, IL-6 injec...

  19. Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys▿

    Sun, Yue; Bailer, Robert T.; Rao, Srinivas S.; Mascola, John R.; Nabel, Gary J.; Koup, Richard A.; Letvin, Norman L.

    2009-01-01

    The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to...

  20. Antibacterial Activity of Recombinant Pig Intestinal Parasite Cecropin P4 Peptide Secreted from Pichia pastoris

    Song, Ki-Duk; Lee, Woon-Kyu

    2014-01-01

    Cecropins (Cec) are antibacterial peptides and their expression is induced in a pig intestinal parasite Ascaris suum by bacterial infection. To explore the usefulness of its activity as an antibiotic, CecP4 cDNA was prepared and cloned into the pPICZ B expression vector and followed by the integration into AOX1 locus in Pichia pastoris. The supernatants from cell culture were collected after methanol induction and concentrated for the test of antimicrobial activity. The recombinant P. patoris...

  1. Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2

    Kogut, Michael; Rothwell, Lisa; Kaiser, Pete

    2002-01-01

    Interleukin-2 (IL-2) exercises an array of biological effects on many cells including the functional activation of cells of the innate immune response. Heterophils, the avian equivalent of the neutrophil, function as professional phagocytes to aid in regulation of innate host defenses. The objective of the present studies was to examine the effects of recombinant chicken IL-2 (rChIL-2) on functional activities of heterophils from chickens during the first 3 weeks after hatch. Peripheral blood...

  2. Anti-HBV activity of TRL mediated by recombinant adenovirus

    Wei-Dong Gong; Ya Zhao; Jun Yi; Jin Ding; Jun Liu; Cai-Fang Xue

    2005-01-01

    AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGlox(delta)E1,3Cre to acquire RAd/TRL, TR, HBVcand hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs 1.60±0.47, P = 0.0266, <0.05) andother control groups (0.63±0.14 vs 8.50±2.78, 8.25±2.26,8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assaysuggested that there were no significant differences in cell metabolic activity between groups (P>0.05).CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

  3. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis. PMID:15177278

  4. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Sepehrizadeh Z; Tabatabaei Yazdi M; Zarrini GH; Hashemi Bozchlou S; Khoshakhlagh P

    2008-01-01

    Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml) were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by ...

  5. Radiative recombination from dark excitons in nanocrystals: Activation mechanisms and polarization properties

    Rodina, Anna V.; Efros, Alexander L.

    2016-04-01

    We analyze theoretically physical mechanisms responsible for the radiative recombination of the ground optically passive ("dark") exciton (DE), which dominates in photoluminescence (PL) of colloidal nanocrystals (NCs) at low temperatures. The DE becomes optically active due to its mixing with the bright excitons caused by an external magnetic field, dangling-bond spins or by acoustic and optical phonons. These activation mechanisms mix the DE with different bright excitons and, consequently, lead to different PL polarization properties, because they are determined by dipole orientations of the bright excitons, which the DE is coupled with. We show that the PL polarization properties of prolate and oblate shape NCs are different due to different activation mechanisms responsible for the DE recombination.

  6. Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba.

    Pabel, Christian T; Vater, Joachim; Wilde, Christopher; Franke, Peter; Hofemeister, Jürgen; Adler, Barbara; Bringmann, Gerhard; Hacker, Jörg; Hentschel, Ute

    2003-01-01

    The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites characteristic of Aplysina aerophoba. For this purpose, bacterial isolation was performed on agar plates to which sponge tissue extract had been added. Following screening for antifungal and antimicrobial activities, 5 strains were chosen for more detailed analyses. 16S ribosomal DNA sequencing revealed that all isolates belonged to the genus Bacillus, specifically B. subtilis and B. pumilus. Using a combination of matrix-assisted laser desorption/ ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference strains, the bioactive metabolites were identified as lipopeptides. PMID:14730425

  7. The role of p53 in radiation activated recombination in human teratocarcinoma cells

    Purpose/Objective: We have previously demonstrated that ionizing radiation can activate a DNA recombination pathway in mammalian cells. In this project, we investigated the role of p53 in radiation activated recombination in ovarian tumor cell lines, and also the effect of p53 status on radiation sensitivity in this cell system. Materials and Methods: PA-1 teratocarcinoma cells, which express wild type p53, were transfected with an HPV16 E6 expression vector (PA-1/E6) which promotes p53 degradation, or transfected with a similar vector coding only for the neomycin phosphotransferase gene (PA-1/Neo). Approximately 3 weeks after this transfection, surviving cells were pooled and expanded. Nuclear extracts were made from each cell line three hours after cells were irradiated with doses ranging from 0 Gy to 10 Gy. Briefly, cells were lysed in sucrose buffer, and the nuclei and cytoplasm separated by centrifugation. Nuclei were lysed in low salt buffer followed by high salt buffer and centrifugation (as described by Johnson et al., Biotechniques 19:193-5, 1995). The ability of these nuclear extracts to rejoin or recombine EcoRI linearized pSV2neo was then determined. The effect of irradiation and P53 on stable transfection determined by assessing transfection of a Hygromycin marker vector (pSV2HPH). Radiation sensitivity was also determined. Results: Nuclear extracts from unirradiated cells had demonstrated end joining activity. PA-1/Neo had little end joining activity as measured by dimerization of linearized pSV2neo. Recutting of these dimers with EcoRI almost completely removed the dimer. PA-1/E6 demonstrated significantly more dimer formation (∼10 fold more) than PA-1/Neo. These dimers could only be reduced to ∼50% of PA-1/E6 control by redigestion with EcoRI. Nuclear extracts generated 3 hours after irradiation also had end joining activity. After 10Gy, PA-1/Neo demonstrated markedly elevated end joining activity to the level seen in unirradiated PA-1/E6. This

  8. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

  9. Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus)

    Xianlei Wang; Xungang Tan; Pei-Jun Zhang; Yuqing Zhang; Peng Xu

    2014-12-01

    During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month-old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

  10. The use of recombinant activated coagulation factor VII for spine surgery

    Weiskopf, Richard B.

    2004-01-01

    This article focuses on our current understanding of the role of activated coagulation factor VII (FVIIa) in coagulation, the current evidence regarding the efficacy and safety of recombinant FVIIa (rFVIIa), and thoughts regarding the use of rFVIIa in spine surgery. rFVIIa is approved in many countries (including the European Union and the USA) for patients with hemophilia and inhibitors (antibodies) to coagulation factors VIII or IX. High circulating concentrations of FVIIa, achieved by exog...

  11. Study of Desorption Agent for LADS Process

    Zhang Xiaojing; Qin Ruyi; Liu Jinlong

    2003-01-01

    This article refers to the procedure for selection, evaluation and development of the LADS-D desorption agent associated with the LADS-A adsorbent used in the non-hydroprocessing adsorptive desulfurization (LADS) process for FCC naphtha developed by LPEC Refining Research Institute. The LADS-D desorption agent can effectively remove the sulfides adsorbed on the LADS-A adsorbent. The saturated LADS-A adsorbent can be instantly regenerated by the LADS-D desorption agent to recover its adsorption activity. The LADS-D desorption agent can not only effectively remove all impurities adsorbed on the adsorbent, but also has strong ability to dissolve the impurities to keep a stable desorption efficiency of adsorbent to be basically commensurate with fresh adsorbent after extended use.

  12. Thermal desorption study of physical forces at the PTFE surface

    Thermal desorption spectroscopy (TDS) of the polytetrafluoroethylene (PTFE) surface was successfully employed to study the possible role of physical forces in the enhancement of metal-PTFE adhesion by radiation. The thermal desorption spectra were analyzed without assumptions to yield the activation energy for desorption over a range of xenon coverage from less than 0.1 monolayer to more than 100 monolayers. For multilayer coverage, the desorption is zero-order with an activation energy equal to the sublimation energy of xenon. For submonolayer coverages, the order for desorption from the unirradiated PTFE surface is 0.73 and the activation energy for desorption is between 3.32 and 3.36 kcal/mol; less than the xenon sublimation energy. The effect of irradiation is to increase the activation energy for desorption to as high as 4 kcal/mol at low coverage. 13 references

  13. Thermal desorption study of physical forces at the PTFE surface

    Thermal desorption spectroscopy (TDS) of the polytetrafluoroethylene (PTFE) surface was successfully employed to study the possibile role of physical forces in the enhancement of metal-PTFE adhesion by radiation. The thermal desorption spectra were analyzed without assumptions to yield the activation energy for desorption over a range of xenon coverage from less than 0.1 monolayer to more than 100 monolayers. For multilayer coverage, the desorption is zero-order with an activation energy equal to the sublimation energy of xenon. For submonolayer coverages, the order for desorption from the unirradiated PTFE surface is 0.73 and the activation energy for desorption is between 3.32 and 3.36 kcal/mol; less than the xenon sublimation energy. The effect of irradiation is to increase the activation energy for desorption to as high as 4 kcal/mol at low coverage

  14. Thermal desorption study of physical forces at the PTFE surface

    Wheeler, D. R.; Pepper, S. V.

    1987-01-01

    Thermal desorption spectroscopy (TDS) of the polytetrafluoroethylene (PTFE) surface was successfully employed to study the possible role of physical forces in the enhancement of metal-PTFE adhesion by radiation. The thermal desorption spectra were analyzed without assumptions to yield the activation energy for desorption over a range of xenon coverage from less than 0.1 monolayer to more than 100 monolayers. For multilayer coverage, the desorption is zero-order with an activation energy equal to the sublimation energy of xenon. For submonolayer coverages, the order for desorption from the unirradiated PTFE surface is 0.73 and the activation energy for desorption is between 3.32 and 3.36 kcal/mol; less than the xenon sublimation energy. The effect of irradiation is to increase the activation energy for desorption to as high as 4 kcal/mol at low coverage.

  15. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

    Grishina, Zoryana; Ostrowska, Ewa; Halangk, Walter; Sahin-Tóth, Miklós; Reiser, Georg

    2005-01-01

    Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca2...

  16. Construction and expression of a recombinant antibody-targeted plasminogen activator

    Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

  17. Activation and block of recombinant GABAA receptors by pentobarbitone: a single-channel study

    Akk, Gustav; Steinbach, Joe Henry

    2000-01-01

    Recombinant GABAA receptors (α1β2γ2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell-attached single-channel patch clamp.Clusters of single-channel activity elicited by 500 μM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel op...

  18. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

  19. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.

    2016-01-01

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  20. Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination.

    Huang, Shengfeng; Tao, Xin; Yuan, Shaochun; Zhang, Yuhang; Li, Peiyi; Beilinson, Helen A; Zhang, Ya; Yu, Wenjuan; Pontarotti, Pierre; Escriva, Hector; Le Petillon, Yann; Liu, Xiaolong; Chen, Shangwu; Schatz, David G; Xu, Anlong

    2016-06-30

    Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon. PMID:27293192

  1. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks.

    Minca, Eugen C; Kowalski, David

    2010-06-11

    DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. PMID:20541998

  2. Cre-dependent DNA recombination activates a STING-dependent innate immune response.

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W Samantha N; Cain, Jason E; Behlke, Mark A; Gough, Daniel J; G Williams, Bryan R; Hornung, Veit; Gantier, Michael P

    2016-06-20

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  3. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

    Bo Sun; Zhao-Shen Li; Zhen-Xing Tu; Guo-Ming Xu; Yi-Qi Du

    2006-01-01

    AIM: To construct a live attenuated Salmonella typhimurium (S.typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity.METHODS: By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments.RESULTS: A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recompinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylor(i) whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response.CONCLUSION: The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

  4. Stability of Recombinant Tissue Plasminogen Activator at −30 °C Over One Year

    Alkatheri, Abdulmalik

    2012-01-01

    Recombinant tissue plasminogen activator (rt-PA) is used to restore patency and avoid inadvertent removal of peripheral and central venous catheters. rt-PA was reconstituted (1 mg/mL) then cryopreserved at −30 °C for 1, 2, 3, 6, 8, and 12 months and, then its stability was determined. After cryopreservation for one and two months, rt-PA kept more than 95% of its activity compared to standard samples, while cryopreservation for three months caused 8% loss of activity. However, after cryopreservation for six months or more, rt-PA retained only 87.5% or less activity compared to standard samples. Therefore, it is recommended that reconstituted rt-PA be cryopreserved at −30 °C for a maximum period of three months. PMID:24275785

  5. Immunostimulatory Activity of Recombinant Mycobacterium bovis BCG That Secretes Major Membrane Protein II of Mycobacterium leprae

    Makino, Masahiko; Maeda, Yumi; Inagaki, Katsuya

    2006-01-01

    We previously demonstrated that major membrane protein II (MMP-II) is one of the immunodominant antigens (Ags) of Mycobacterium leprae capable of activating T cells through Toll-like receptor 2. Based on the observation that Mycobacterium bovis BCG secreting a 30-kDa protein offered better protection against tuberculosis, we constructed a recombinant BCG strain (BCG-SM) that secretes MMP-II to improve the potency of BCG against leprosy. The secreted MMP-II protein from BCG-SM stimulated monoc...

  6. Does intravenous administration of recombinant tissue plasminogen activator for ischemic stroke can cause inferior myocardial infarction?

    Mostafa Almasi

    2016-06-01

    Full Text Available Recombinant tissue plasminogen activator (rTPA is one of the main portions of acute ischemic stroke management, but unfortunately has some complications. Myocardial infarction (MI is a hazardous complication of administration of intravenous rTPA that has been reported recently. A 78-year-old lady was admitted for elective coronary artery bypass graft surgery. On the second day of admission, she developed acute left hemiparesis and intravenous rTPA was administered within 120 minutes. Three hours later, she has had chest pain. Rescue percutaneous coronary intervention was performed on right coronary artery due to diagnosis of inferior MI, and the symptoms were resolved.

  7. Characterization of the Active Surface Species Responsible for UV-Induced Desorption of O2 from the Rutile TiO2(110) Surface

    Henderson, Michael A.; Shen, Mingmin; Wang, Zhitao; Lyubinetsky, Igor

    2013-03-21

    We have examined the chemical and photochemical properties of molecular oxygen on the (110) surface of rutile TiO2 at 100 K using electron energy loss spectroscopy (EELS), photon stimulated desorption (PSD) and scanning tunneling microscopy (STM). Oxygen chemisorbs on the TiO2(110) surface at 100 K through charge transfer from surface Ti3+ sites. The charge transfer process is evident in EELS by a decrease in the intensity of the Ti3+ d-to-d transition in EELS at ~0.9 eV and formation of a new loss ~2.8 eV. Based on comparisons with the available homogeneous and heterogeneous literature for complexed/adsorbed O2, the species responsible for the 2.8 eV peak can be assigned to a surface peroxo (O2 2-) state of O2. This species was identified as the active form of adsorbed O2 on TiO2(110) for PSD. The adsorption site of this peroxo species was assigned to that of a regular five-cooridinated Ti4+ (Ti5c) site based on comparisons between the UV exposure dependent behavior of O2 in STM, PSD and EELS data. Assignment of the active form of adsorbed O2 to a peroxo species at normal Ti5c sites necessitates reevaluation of the simple mechanism in which a single valence band hole neutralizes a singly charged O2 species (superoxo or O2-) leading to desorption of O2 from a physisorbed potential energy surface. This work was supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, and the Chemical Imaging Initiative at Pacific Northwest National Laboratory (PNNL), and was performed in the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the DOE's Office of Biological and Environmental Research and located at PNNL.

  8. Expression, Purification and Activity Assay of Two New Recombinant Antagonists of Fibrinogen Receptor

    Jianbo Yang

    2005-01-01

    Full Text Available The gene sequence of Decorsin which is extracted from a kind of North American leeches was synthesized. Two recombinant proteins, Annexin V plus Decorsin (AnnV-D39 and Annexin V plus the carboxyl terminal 27 amino acid residues of Decorsin(AnnV-D27, were constructed. And a 10 amino acids linker peptide of GGGGSGGGGS was inserted between Annexin V and Decorsin in AnnV-D39. Using pET-28(a+ as an expressing vector, both two recombinant proteins were expressed in E. Coli BL21(DE3 with high efficiency as inclusion bodies. The expression products were purified by DEAE-Cellulose 52 and Sepharose CL-4B chromatography under denaturing condition. Platelet Aggregation Assay (PAA shows that AnnV-D39 has good anti-platelet aggregation activity. However, AnnV-D27 shows no such activities in any PAA test. AnnV-D39 shows good anti-platelet aggregation activity as a new antagonist of fibrinogen receptor, while Annv-D27 needs re-modification

  9. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  10. Technetium-99m-labeled recombinant tissue plasminogen activator for the imaging of emboli in vivo

    Takahashi, Akihiro; Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Kamiyama, Hiroyasu; Abe, Hiroshi (Hokkaido Univ., Sapporo (Japan). School of Medicine)

    1993-07-01

    Tissue-type plasminogen activator (t-PA) effectively lyses activate thrombus by direct action. Recombinant t-PA (rt-PA) was labeled with technetium-99m ([sup 99m]Tc) to investigate the in vivo binding to fibrin clots in a feline cerebral embolism model created by insertion of an artificial fibrin clot within the carotid artery. [sup 99m]Tc-rt-PA administered intravenously provided clearer imaging of clots after priming with cold rt-PA, with uptake peaking 5-10 minutes after the injection. [sup 99m]Tc-labeled human serum albumin was not retained at clot sites. Systemically administered [sup 99m]Tc-rt-PA binds to fibrin clots within carotid arteries in our feline model. Our results suggest that the interaction of intrinsic plasminogen activator inhibitors with extrinsically administered rt-PA may regulate the demonstration of a clot, although the precise mechanism is unclear. (author).

  11. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast

    Liu, Bo; Shi, DanYang; Chang, Shaohong; Gong, Xin; Yu, YunZhou; Sun, Zhiwei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. ...

  12. Expression, Purification and Activity Assay of the Recombinant Protein of Catechol-O-Methyltransferase from Chinese White Shrimp (Fenneropenaeus chinensis

    Dian-Xiang Li

    2010-01-01

    Full Text Available Problem statement: We have previously cloned a gene of Chinese white shrimp Catechol O-Methyltransferase (designated Fc-COMT and characterized the gene expression pattern. In this study, expression and purification as well as activity assay of the recombinant Fc-COMT was further conducted. Approach: Using pET-30a (+ as a prokaryotic expression vector, the recombinant Fc- COMT was expressed in the supernatant of Escherichia coli lysate and easily purified by His-Bind resin chromatography. SDS-PAGE analysis showed that the molecular mass of recombinant Fc-COMT was approximately 30,000 Da, in good agreement with the software-predicted molecular weight. The enzymatic activity of recombinant Fc-COMT was tested using Dihydroxybenzoic Acid (DHBAc as a substrate. Results: The methyl products of DHBAc, Vanillic Acid (VA and Isovanillic Acid (IVA, were detected in the enzymatic reaction mixture with recombinant Fc-COMT by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS. Conclusion: The recombinant Fc-COMT has catalytic activity of transferring methyl group from S-Adenosyl-L-Methionine (SAM to the 3’ hydroxyl or 4’ hydroxyl group of benzyl ring of DHBAc.

  13. Adsorption isotherms, kinetics, thermodynamics and desorption studies of 2,4,6-trichlorophenol on oil palm empty fruit bunch-based activated carbon

    The adsorption characteristics of 2,4,6-trichlorophenol (TCP) on activated carbon prepared from oil palm empty fruit bunch (EFB) were evaluated. The effects of TCP initial concentration, agitation time, solution pH and temperature on TCP adsorption were investigated. TCP adsorption uptake was found to increase with increase in initial concentration, agitation time and solution temperature whereas adsorption of TCP was more favourable at acidic pH. The adsorption equilibrium data were best represented by the Freundlich and Redlich-Peterson isotherms. The adsorption kinetics was found to follow the pseudo-second-order kinetic model. The mechanism of the adsorption process was determined from the intraparticle diffusion model. Boyd plot revealed that the adsorption of TCP on the activated carbon was mainly governed by particle diffusion. Thermodynamic parameters such as standard enthalpy (ΔHo), standard entropy (ΔSo), standard free energy (ΔGo) and activation energy were determined. The regeneration efficiency of the spent activated carbon was high, with TCP desorption of 99.6%.

  14. Activity and stability of recombinant human superoxide dismutase in buffer solutions and hypothermic perfusates.

    Senoo,Yoshimasa

    1988-06-01

    Full Text Available The stability of recombinant human superoxide dismutase (r-hSOD in buffer solutions was studied in solutions at various pH and temperatures. Additionally, we studied the effects of incubation with proteases, serum and two types of hypothermic perfusates. R-hSOD was stable in the pH range of 6-11 and at temperatures up to 80 degrees C for 30 min. R-hSOD activity was not affected by incubation with trypsin, aminopeptidase M or serum for 2 h. R-hSOD activity determined at various temperatures (4-37 degrees C did not vary remarkably. R-hSOD in hypothermic perfusates was stable at 4-37 degrees C for 24 h.

  15. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo. PMID:27401969

  16. Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.

    Ying-Ming Wang

    Full Text Available Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.

  17. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

    Tan, B H; Fu, J; Sugrue, R J; Yap, E H; Chan, Y C; Tan, Y H

    1996-02-15

    The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA. PMID:8607261

  18. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  19. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

    Wang, Chunhe; Mooney, Jeffery L; Meza-Romero, Roberto; Chou, Yuan K; Huan, Jianya; Vandenbark, Arthur A; Offner, Halina; Burrows, Gregory G

    2003-08-15

    Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells. PMID:12902496

  20. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    Zita Nagy

    2016-02-01

    Full Text Available DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR, a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1 is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ and Homologous Recombination (HR repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose Polymerases (PARPs TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.

  1. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    Nagy, Zita; Kalousi, Alkmini; Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-02-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  2. Failure of Recombinant Activated Factor VII in Treatment of Diffuse Alveolar Hemorrhage due to Cryoglobulinemic Vasculitis

    Dania Khoulani

    2014-01-01

    Full Text Available Diffuse alveolar hemorrhage (DAH is a serious complication of the small vessel vasculitis syndromes and carries a high mortality. Recombinant activated factor VII (rFVIIa is used to treat bleeding in patients with hemophilia and antibodies to factor VIII or IX. It is increasingly being used in life-threatening hemorrhage in a variety of other settings in which conventional therapy is unsuccessful. Randomized controlled trials of rFVIIa in DAH are lacking. However, several case reports have described a complete or sustained control of DAH using rFVIIa after patients failed to respond to medical treatment. There are no case reports in the literature describing the use or the failure of rFVIIa in DAH associated with cryoglobulinemic vasculitis. We here report the failure of rFVIIa to control DAH in a patient with CD5+ B-cell non-Hodgkin’s lymphoma and cryoglobulinemic vasculitis.

  3. Annealing effects on recombinative activity of nickel at direct silicon bonded interface

    Takuto Kojima

    2015-09-01

    Full Text Available By performing capacitance transient analyses, the recombination activity at a (110/(100 direct silicon bonded (DSB interface contaminated with nickel diffused at different temperatures, as a model of grain boundaries in multicrystalline silicon, was studied. The trap level depth from the valence band, trap density of states, and hole capture cross section peaked at an annealing temperature of 300 °C. At temperatures ⩾400 °C, the hole capture cross section increased with temperature, but the density of states remained unchanged. Further, synchrotron-based X-ray analyses, microprobe X-ray fluorescence (μ-XRF, and X-ray absorption near edge structure (XANES analyses were performed. The analysis results indicated that the chemical phase after the sample was annealed at 200 °C was a mixture of NiO and NiSi2.

  4. Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

    The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

  5. First-principles study of water desorption from montmorillonite surface.

    Zhang, Yao; Meng, Yingfeng; Liu, Houbin; Yang, Mingli

    2016-05-01

    Knowledge about water desorption is important to give a full picture of water diffusion in montmorillonites (MMT), which is a driving factor in MMT swelling. The desorption paths and energetics of water molecules from the surface of MMT with trapped Li(+), Na(+) or K(+) counterions were studied using periodic density functional theory calculations. Two paths-surface and vacuum desorption-were designed for water desorption starting from a stationary structure in which water bonds with both the counterion and the MMT surface. Surface desorption is energetically more favorable than vacuum desorption due to water-surface hydrogen bonds that help stabilize the intermediate structure of water released from the counterion. The energy barriers of water desorption are in the order of Li(+) > Na(+) > K(+), which can be attributed to the short ionic radius of Li(+), which favors strong binding with the water molecule. The temperature dependence of water adsorption and desorption rates were compared based on the computed activation energies. Our calculations reveal that the water desorption on the MMT surface has a different mechanism from water adsorption, which results from surface effects favoring stabilization of water conformers during the desorption process. PMID:27083565

  6. Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi

    The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with 111In. The rt-PA was coupled with the cyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with 111In bound exlusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5-1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot:blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with 111In without destroying the ability of the protein to loacalize n clot and images of forming clot can be obtained with this agent within 1 h after administration. (orig.)

  7. Recombinant activated factor Ⅶ in hemophagocytic lymphohistiocytosis with disseminated intravascular coagulation

    ZHUANG Jun-ling; JIANG Qing-wei; XU Ying; WANG Shu-jie

    2011-01-01

    Hemophagocytic lymphohistiocytosis (HLH) is a lifethreatening disorder due to hyperinflammation resulting in infiltration of different organs with extensive hemophagocytosis. Severe coagulopathy was one of the main reasons for death in HLH. Over secretion of plasminogen activator by activated macrophages leads to hyperfibrinolysis. We reported a 36-year-old woman who was diagnosed as HLH probably secondary to lymphoma. Massive bleeding from gut and retroperitoneal area were not able to be controlled by conventional hemostatic treatments. This patient received one dose recombinant activated factor Ⅶ (rFVlla) 3.6 mg (70 μg/kg). Hemostatic effect was achieved in 0.5 hour and lasted 24 hours. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were quickly corrected to normal ranges.Fibrinogen level elevated from 0.5 g/L before using rFVIla to 1.8 g/L 20 hours after. Although dexamethasone and etopside were administrated to treat HLH, this patient died from septic shock after persistent neutropenia. This suggests that rFVlla may be effective in the management of intractable hemorrhage in patients with HLH.

  8. Bimolecular recombination reactions: K-adiabatic and K-active forms of the bimolecular master equations and analytic solutions

    Ghaderi, Nima

    2016-03-01

    Expressions for a K-adiabatic master equation for a bimolecular recombination rate constant krec are derived for a bimolecular reaction forming a complex with a single well or complexes with multiple well, where K is the component of the total angular momentum along the axis of least moment of inertia of the recombination product. The K-active master equation is also considered. The exact analytic solutions, i.e., the K-adiabatic and K-active steady-state population distribution function of reactive complexes, g(EJK) and g(EJ), respectively, are derived for the K-adiabatic and K-active master equation cases using properties of inhomogeneous integral equations (Fredholm type). The solutions accommodate arbitrary intermolecular energy transfer models, e.g., the single exponential, double exponential, Gaussian, step-ladder, and near-singularity models. At the high pressure limit, the krec for both the K-adiabatic and K-active master equations reduce, respectively, to the K-adiabatic and K-active bimolecular Rice-Ramsperger-Kassel-Marcus theory (high pressure limit expressions). Ozone and its formation from O + O2 are known to exhibit an adiabatic K. The ratio of the K-adiabatic to the K-active recombination rate constants for ozone formation at the high pressure limit is calculated to be ˜0.9 at 300 K. Results on the temperature and pressure dependence of the recombination rate constants and populations of O3 will be presented elsewhere.

  9. Master Equation for Hydrogen Recombination on Grain Surfaces

    Biham, O; Pirronello, V; Vidali, G; Biham, Ofer; Furman, Itay; Pirronello, Valerio; Vidali, Gianfranco

    2000-01-01

    Recent experimental results on the formation of molecular hydrogen on astrophysically relevant surfaces under conditions similar to those encountered in the interstellar medium provided useful quantitative information about these processes. Rate equation analysis of experiments on olivine and amorphous carbon surfaces provided the activation energy barriers for the diffusion and desorption processes relevant to hydrogen recombination on these surfaces. However, the suitability of rate equations for the simulation of hydrogen recombination on interstellar grains, where there might be very few atoms on a grain at any given time, has been questioned. To resolve this problem, we introduce a master equation that takes into account both the discrete nature of the H atoms and the fluctuations in the number of atoms on a grain. The hydrogen recombination rate on microscopic grains, as a function of grain size and temperature, is then calculated using the master equation. The results are compared to those obtained fro...

  10. The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

    WANG An-ping; SUN Huai-chang; WANG Jian-ye; WANG Yong-juan; YUAN Wei-feng

    2007-01-01

    To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

  11. The role of beaded activated carbon's pore size distribution on heel formation during cyclic adsorption/desorption of organic vapors.

    Jahandar Lashaki, Masoud; Atkinson, John D; Hashisho, Zaher; Phillips, John H; Anderson, James E; Nichols, Mark

    2016-09-01

    The effect of activated carbon's pore size distribution (PSD) on heel formation during adsorption of organic vapors was investigated. Five commercially available beaded activated carbons (BAC) with varying PSDs (30-88% microporous) were investigated. Virgin samples had similar elemental compositions but different PSDs, which allowed for isolating the contribution of carbon's microporosity to heel formation. Heel formation was linearly correlated (R(2)=0.91) with BAC micropore volume; heel for the BAC with the lowest micropore volume was 20% lower than the BAC with the highest micropore volume. Meanwhile, first cycle adsorption capacities and breakthrough times correlated linearly (R(2)=0.87 and 0.93, respectively) with BAC total pore volume. Micropore volume reduction for all BACs confirmed that heel accumulation takes place in the highest energy pores. Overall, these results show that a greater portion of adsorbed species are converted into heel on highly microporous adsorbents due to higher share of high energy adsorption sites in their structure. This differs from mesoporous adsorbents (low microporosity) in which large pores contribute to adsorption but not to heel formation, resulting in longer adsorbent lifetime. Thus, activated carbon with high adsorption capacity and high mesopore fraction is particularly desirable for organic vapor application involving extended adsorption/regeneration cycling. PMID:27173087

  12. Recombinant expression, antimicrobial activity and mechanism of action of tritrpticin analogs containing fluoro-tryptophan residues.

    Arias, Mauricio; Hoffarth, Elesha R; Ishida, Hiroaki; Aramini, James M; Vogel, Hans J

    2016-05-01

    The increase in antibiotic-resistant bacterial infections has prompted significant academic research into new therapeutic agents targeted against these pathogens. Antimicrobial peptides (AMPs) appear as promising candidates, due their potent antimicrobial activity and their ubiquitous presence in almost all organisms. Tritrpticin is a member of this family of peptides and has been shown to exert a strong antimicrobial activity against several bacterial strains. Tritrpticin's main structural characteristic is the presence of three consecutive Trp residues at the center of the peptide. These residues play an important role in the activity of tritrpticin against Escherichia coli. In this work, a recombinant version of tritrpticin was produced in E. coli using calmodulin as a fusion protein expression tag to overcome the toxicity of the peptide. When used in combination with glyphosate, an inhibitor of the endogenous synthesis of aromatic amino acids, this expression system allowed for the incorporation of fluorinated Trp analogs at very high levels (>90%). The antimicrobial activity of the 4-, 5- and 6-fluoro-Trp-containing tritrpticins against E. coli was as strong as the activity of the native peptide. Similarly, the tritrpticin analogs exhibited comparable abilities to perturb and permeabilize synthetic lipid bilayers as well as the outer and inner membrane of E. coli. Furthermore, the use of 19F NMR spectroscopy established that each individual fluoro-Trp residue interacts differently with SDS micelles, supporting the idea that each Trp in the original tritrpticin plays a different role in the perturbing/permeabilizing activity of the peptide. Moreover, our work demonstrates that the use of fluoro-Trp in solvent perturbation 19F NMR experiments provides detailed site-specific information on the insertion of the Trp residues in biological membrane mimetics. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai

  13. Active immunotherapy of allergic asthma with a recombinant human interleukin-5 protein as vaccine in a murine model

    TAN Guang-hong; WANG Cai-chun; HUANG Feng-ying; WANG Hua; HUANG Yong-hao; LIN Ying-ying

    2007-01-01

    Background Eosinophils are highly related to allergic asthma inflammation. Interleukin (IL)-5 is the major chemokine of eosinophils, inhibition of the activity of IL-5 thus seems to be a potential approach to asthma therapy. The current study was performed to determine whether a recombinant human IL-5 protein as a xenogeneic vaccine has the capability of inducing anti-asthma activities.Methods Recombinant human IL-5 was used as a protein vaccine. Mouse asthma model was established to observe the anti-asthma activities. Lung histology was observed; eosinophils in blood and bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness was determined by whole body plethysmograph. Antibody characters and cytokines were detected with enzyme linked immunosorbent assay (ELISA) and Western blot assay.Results Vaccination with recombinant human IL-5 protein as vaccine significantly reduced airway inflammation and airway hyperresponsiveness, and shifted the cytokine production from Th2 (IL-4) to Th1 (INF-γ) in mice allergic-asthma model. Immunization with recombinant human IL-5 protein vaccine bypassed the immunological tolerance and induced production of polyclonal antibodies that were cross-reactive with murine IL-5.Conclusions Active immunization with xenogeneic homologous IL-5 may be a possible therapeutic approach to the treatment of asthma and potentially of other eosinophilic disorders.

  14. Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

    Janke, M.; Peeters, B.; Zhao, H.; Leeuw, O.; Moormann, R.J.M.; Arnold, A.; Ziouta, Y.; Fournier, P.; Schirrmacher, V.

    2008-01-01

    A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was prod

  15. Treatment of massive gastrointestinal bleeding occurred during autologous stem cell transplantation with recombinant activated factor VII and octreotide

    Erman Atas

    2015-01-01

    Full Text Available After hematopoietic stem cell transplantation (HSCT, patients may suffer from bleeding. One of the bleeding type is gastrointestinal (GI which has serious morbidity and mortality in children with limited treatment options. Herein, we presented a child with upper GI bleeding post autologous HSCT controlled successfully by using recombinant activated factor VII (rFVIIa and octreotide infusion.

  16. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  17. Adsorption onto Activated Carbon Fiber Cloth and Electrothermal Desorption of Volatile Organic Compound (VOCs): A Specific Review

    Pierre Le Cloirec

    2012-01-01

    A general research program, focusing on activated carbon fiber cloths (ACFC) and felt for environmental protection was performed. The objectives were multiple: (i) a better understanding of the adsorption mecha- nisms of these kinds of materials; (ii) the specification and optimization of new processes using these adsorbents; (iii) the modeling of the adsorption of organic pollutants using both the usual and original approaches; (iv) applications of ACFC in industrial processes. The general question was: how can activated carbon fiber cloths and felts be used in air treatment processes for the protection of environment. In order to provide an answer, different approaches were adopted. The materials (ACFC) were characterized in terms of macro structure and internal porosity. Specific studies were performed to get the air flow pattern through the fabrics. Head loss data were generated and modeled as a fi.mction of air velocity. The performances of ACF to remove volatile organic compounds (VOCs) were approached with the adsorption isotherms and breakthrough curves in various operating conditions. Regenera- tion by Joule effect shows a homogenous heating of adsorber modules with rolled or pleated layers. Examples of industrial developments were presented showing an interesting technology for the removal of VOCs, such as dichloromethane, benzene, isopropyl alcohol and toluene, alone or in a complex mixture.

  18. [Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect].

    Pokrovskaia, M V; Pokrovskiĭ, V S; Aleksandrova, S S; Anisimova, N Iu; Adrianov, R M; Treshchalina, E M; Ponomarev, G V; Sokolov, N N

    2013-01-01

    The recombinant producer of Rhodospirillum rubrum L-asparaginase (RrA) was received and purification procedure of RrA was developed. It was shown that RrA has following biochemical and catalytic characteristics: K(m) for L-asn 0.22 MM, pH optimum 9.2; temperature optimum 54 degrees C; pI = 5.1 +/- 0.3; L-gln activity seems to be low-to-negligible. K562, DU145 and MDA-MB-231 cellular lines displayed significant sensitivity towards the enzyme (IC50 = 1.80; 9.19 and 34.62 ME/ml, respectively. In comparison with L-asparaginases from E. coli II type (EcA) and Erwinia carotovora (EwA) cytotoxicity of RrA seems to be higher than EwA, but lower than EcA. 10-fold i.p. RrA administration (4000 ME/kg per day) in L5178y bearing mice showed T/C = 172%. The received results show that RrA belongs to I type cellular L-asparaginases with low L-gln activity and the high antiproliferative effect. PMID:23789346

  19. Occupational exposure to complex mixtures of volatile organic compounds in ambient air: desorption from activated charcoal using accelerated solvent extraction can replace carbon disulfide?

    Fabrizi, Giovanni; Fioretti, Marzia; Rocca, Lucia Mainero

    2013-01-01

    A desorption study of 57 volatile organic compounds (VOCs) has been conducted by use of accelerated solvent extraction (ASE) and gas chromatography-mass spectrometry. Different solvents were tested to extract activated charcoal tubes with the objective of replacing carbon disulfide, used in official methods, because of its highly toxic health and environmental effects. Extraction conditions, for example temperature and number of cycles, were investigated and optimized. The definitive extraction procedure selected was use of acetone at 150 °C and two consecutive extraction cycles at a pressure of 1,500 psi. Considering a sample volume of 0.005 Nm(3), corresponding to a sampling time of 8 h at a flow rate of 0.01 L min(-1), the method was validated over the concentration range 65-26,300 μg Nm(-3). The lowest limit of quantification was 6 μg Nm(-3), and recovery for the 93 % of analytes ranged from 65 to 102 %. For most of the compounds, relative standard deviations were less than 15 % for inter and intra-day precision. Uncertainty of measurement was also determined: the relative expanded uncertainty was always below 29.6 %, except for dichlorodifluoromethane. This work shows that use of friendlier solvent, for example acetone, coupled with use of ASE, can replace use of CS(2) for chemical removal of VOCs from activated charcoal. ASE has several advantages over traditional solvent-extraction methods, including shorter extraction time, minimum sample manipulation, high reproducibility, and less extraction discrimination. No loss of sensitivity occurs and there is also a salutary effect on bench workers' health and on the smell of laboratory air. PMID:22968683

  20. Activity of Recombinant Human Interleukin-15 against Tumor Recurrence and Metastasis in Mice

    Feng Tang; Luting Zhao; Yan Jiang; Denian Ba; Lianxian Cui; Wei He

    2008-01-01

    Transplantable experimental tumor models were constructed to study the activities of recombinant human interleukin-15 (rhIL-15) against tumor recurrence and metastasis. The results showed that tumor nodule formation was retarded and tumor growth was inhibited in the subcutaneous tumor model of LA795 lung adenocarcinoma after treatment with rhIL-15, and the survival rate of T739 tumor-bearing mice treated with rhIL-15 was much higher than that of mice treated with either saline or with the same dose of rhIL-2. This indicats that rhIL-15 had better antitumor effect than rhIL-2 at the same dose level. In some rhIL-15 treated mice, the tumor cells inoculated subcutaneously were eradicated and there was no tumor formation even 138 days after tumor cell inoculation. The tumor-free mice were rechallenged with live tumor cells and no tumor reoccurred in the following two months in all of these mice, indicating that long-lasting antitumor systemic immunity developed. It was also shown that tumor recurrence and metastasis were inhibited markedly after treatment with rhIL-15, but not with the same dose of rhIL-2, in both subcutaneously and intravenously disseminated tumor models of LA795 lung adenocarcinoma. Simultaneously, the CTL and NK cell activities of the splenocytes obtained from tumor-bearing mice that had been treated with either rhIL-15 or rhIL-2 were both markedly enhanced. However, the enhancement of CTL and NK cell activities was more significant in rhIL-15 treated mice than that in rhIL-2 treated mice. This suggests that the anti-tumor effect of rhIL-15 in vivo was achieved by enhancing the CTL and NK cell activities in tumor immune response. Cellular & Molecular Immunology. 2008;5(3):189-196.

  1. Recombinant-activated factor Ⅶ and neuronal apoptosis in a rat model of intracerebral hemorrhage

    Qiang Li; Wei Li; Suju Ding; Jianping Tang; Jing Fang; Benqiang Deng; Tao Wu

    2009-01-01

    BACKGROUND:Activated clotting factor Ⅶ has been demonstrated to exhibit obvious anti-apoptosis effects.OBJECTIVE:To observe the effect of activated clotting factor Ⅶ on neuronal apoptosis at different time points following rat intracerebral hemorrhage (ICH).DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Neurobiological Laboratory of Second Military Medical University from October 2005 to April 2006.MATERIALS:Recombinant-activated clotting factor Vlla (rFⅧa) was purchased from Danish Novo Nordisk,Denmark.In situ cell death detection kit-POD kit was purchased from Roche,Switzerland.Caspase-3 activity determination kit from Biovision,USA.METHODS:A total of 72 healthy,male,Sprague Dawley rats,aged 5-8 months,were randomly assigned to three groups (n=24):sham-operated,ICH model,and rFⅧa.In the ICH model and rFⅧa groups,80.0 μL autologous non-clotting blood from rat tails was injected into the right caudate putamen to establish the ICH.The empty microinjector was inserted into the caudate putamen in the sham-operated group.The ICH model and rFⅧa groups were subdivided into four subsets separately:6,24,72 hours and 7 clays following ICH.The rats in the rFⅧa group were injected with 160 μg/kg rFⅧa via the dorsal vein of the penis.MAIN OUTCOME MEASURES:Apoptotic cells were detected in the right caudate putamen by TUNEL;caspase-3 activity by spectrophotometry;and rat neurological function was evaluated by neurological functional impairment scales.RESULTS:Rat neurological function was deteriorated at 24,72 hours,and 7 days following ICH.The TUNEL-positive cells and caspase-3 activity in the right caudate putamen was significantly increased in the ICH rats (P<0.05);rFVlla treatment reduced the number of TUNEL-positive cells and caspase-3 activity in the right caudate putamen (P<0.05),and neurological function was significantly improved (P<0.05).CONCLUSION:rFⅧa was applied within 72 hours after ICH,which reduced

  2. Thermal desorption of PTCDA on Cu(111)

    Organic molecules often show very complex thermal desorption spectra. If there is an ordered structure the activation energy for desorption Edes will decrease for the first few layers because of the decreasing van der Waals interaction of the layers to the substrate. This is especially true for the system 3,4,9,10 perylene-tetracarboxylic-dianhydride on Cu(111). The spectra consist of three different signals which can be attributed to the second layer, the multilayer and a crystal phase. The numerical algorithm sketched in this paper gives a desorption energy of 2.35eV for the second layer and 2.2eV for the multilayer

  3. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    Raheem Ullah; Majid Ali Shah; Soban Tufail; Fouzia Ismat; Muhammad Imran; Mazhar Iqbal; Osman Mirza; Moazur Rhaman

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally ...

  4. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  5. Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile (Crocodylus siamensis).

    Hao, Juan; Li, Yan-Wei; Xie, Ming-Quan; Li, An-Xing

    2012-01-01

    Hepcidin, a cysteine-rich cationic antibacterial peptide, plays an important role in human defense against pathogen infection. However, its role in reptile immune response and whether it is involved in antibacterial immune have not yet been proven. In order to study the antibacterial activity of Crocodylus siamensis hepcidin (Cshepc), a common reptile which lives in topic region of Southeast Asia, a cDNA sequence of Cshepc was cloned, which included an open reading frame (ORF) of 300 bp encoding a 99 amino acid preprohepcidin. Cshepc has eight cysteines formed four conserved disulfide bridges, similarly to that of human's. Sequence analysis showed that Cshepc mature peptide was more conserved than that of preprohepcidin. Tissue expression analysis indicated that Cshepc transcripts were highly expressed in the liver, muscle and heart of C. siamensis. Recombinant expressed hepcidin could significantly inhibit the growth of the Gram-negative bacteria Escherichia coli and Aeromonas sobria as well as the Gram-positive bacterium Staphylococcus aureus, and Bacillus subtilis in vitro, suggesting that Cshepc, like human hepcidin could play a role in the antibacterial function in hosts innate immune response. PMID:22967859

  6. Early intracardiac thrombosis in preterm infants and thrombolysis with recombinant tissue type plasminogen activator

    Ferrari, F; Vagnarelli, F; Gargano, G; Roversi, M; Biagioni, O; Ranzi, A; Cavazzuti, G

    2001-01-01

    OBJECTIVES—To determine the incidence of catheter related thrombosis and to test the efficacy of recombinant tissue type plasminogen activator (rt-PA) in preterm infants.
STUDY DESIGN—From January 1995 to December 1998, echocardiography was performed in the first few days of life in 76 very low birthweight (⩽ 1500 g) infants out of a total of 147 having an umbilical catheter placed. When intracardiac thrombosis was diagnosed, rt-PA infusion was performed.
RESULTS—Four infants (5%) developed an intracardiac thrombosis during the first few days of life. In three of them, rt-PA at a dose of 0.4-0.5 mg/kg in a 20-30 minute bolus led to dissolution of the clot. One patient received a three hour infusion after the bolus, at a dose of 0.1 mg/kg/h, with resolution of the thrombus. No systemic effects were observed after rt-PA infusion.
CONCLUSIONS—Early thrombosis may occur as a complication of umbilical catheterisation in preterm infants; early echocardiographic detection of this disorder allows complete, safe, and rapid lysis with rt-PA.

 PMID:11420328

  7. Factors predicting intracerebral hemorrhage of patients treated with intravenous recombinant tissue plasminogen activator

    The use of recombinant tissue plasminogen activator (rt-PA) was approved in Japan in October 2005, and has had a marked effect on the treatment of patients presenting with acute ischemic stroke. Since the administration of rt-PA might cause intracerebral hemorrhage (ICH) and a poor prognosis, it is necessary to identify predictors of ICH after treatment with rt-PA. In this article, we examined 58 consecutive patients with acute ischemic stroke treated with intravenous rt-PA within 3 hours of symptom onset for 45 months, March 2006 to November 2009. In principle, we evaluated patients before and one day after rt-PA with MRI. We made a retrospective comparison of 21 patients with hemorrhagic change on CT and MRI T2* within 36 hours and 37 patients without hemorrhagic change. The rate of ICH with or without symptoms was increased with a higher National Institutes of Health Stroke Scale (NIHSS) and infarction range, defined by diffusion weighted imaging (DWI) Alberta Stroke Programme Early CT Score (ASPECTS). Major artery occlusion and reperfusion, including partial recanalization in MR angiography (MRA), were taken as factors in the hemorrhage group. In conclusion, DWI ASPECTS and NIHSS were useful predictors of ICH after rt-PA administration. (author)

  8. Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli

    ZHU Qing-yi; GU Xiao-jian; YANG Jin; WANG Jun-hong; TANG Bo; WU Hong-fei

    2008-01-01

    Background Eppin(epididymis protease inhibitor)appears to play an important role in primate fertility.However,the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied.To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases,we developed an Escherichia coli expression system for the expression of biologically actire human Eppin.Methods The human Eppin gene was cloned into PET-28a(+)vector after induction with 0.5 mmol/L isopropy-β-D-thiogalactoside(IPTG)at 26℃ for 4 hours,and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography.Afterwards,six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks.Their sera were collected and polyclonal antibodies against His6-Eppin were purified,all of which were further verified by Western-blot and immunofluorescence analysis.Results About 18.33 mg His6-Eppin was obtained from 1-L flask culture.The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin.Conclusion The purified His6-Eppin protein has biological activity,which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.

  9. Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

    The authors previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[125I]-iodo-2'-deoxyuridine (125IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125IUdR above saline-treated controls, whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation. When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125IUdR uptake

  10. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    Pär I Johansson

    2010-06-01

    Full Text Available Pär I Johansson, Sisse R OstrowskiCapital Region Blood Bank, Section for Transfusion Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, DenmarkBackground: Recombinant activated factor VII (rFVIIa, NovoSeven® was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.Objective: To review the evidence supporting the use of rFVIIa for the treatment of patients with congenital bleeding disorders.Patients and methods: English-language databases were searched in September 2009 for reports of randomized controlled trials (RCTs evaluating the ability of rFVIIa to restore hemostasis in patients with congenital bleeding disorders.Results: Eight RCTs involving 256 hemophilic patients with antibodies against coagulation factors, also known as inhibitors, were identified. The evidence supporting the use of rFVIIa in these patients was weak with regard to dose, clinical setting, mode of administration, efficacy, and adverse events, given the limited sample size of each RCT and the heterogeneity of the studies.Conclusion: The authors suggest that rFVIIa therapy in hemophilic patients with inhibitors should be based on the individual’s ability to generate thrombin and form a clot, and not on the patient’s weight alone. Therefore, assays for thrombin generation, such as whole-blood thromboelastography, have the potential to significantly improve the treatment of these patients.Keywords: hemophilia, inhibitors, coagulation factor VIII, coagulation factor IX, rFVIIa, NovoSeven, FEIBA, hemostasis, RCT

  11. Analysis of the technique Thermal Desorption Spectroscopy (TDS) and its Application for the Characterization of Metal -Hydrogen Systems

    limiting step is the recombination of two hydrogen atoms on the surface of the material. When the surface of the sample is in the a phase (solid solution) the rate limiting step is one of the processes that occurs on the surface of the material: the transfer of a hydrogen atom from the bulk to the surface or the recombination and desorption on the surface. By fitting the spectra we obtain an activation energy equal to 31 n 6 kJ/mol for the b phase, and equal to 35 n 3 kJ/mol for the a phase. We also compare the spectra of the powder with the spectra of the granules, foils and wires. Based on this comparison we analyze the effect on the desorption spectra of the characteristic size and geometry of the samples.Finally, we present a study of hydrogen desorption in the Pd-H system poisoned with sulfur. The poisoning produces as main effects a delay of the absorption and desorption processes without any appreciable loss in the storage capacity of the material.The experimental apparatus and theoretical models developed have been applied to the study of a MH system using samples with different morphology, characteristic size, geometry and surface state. The spectra were measured for different hydrogen concentrations and heating speeds. The results obtained encourage the utilization of TDS for the study and characterization of hydrogen desorption in MH systems. The technique is suited for the detailed analysis of the physical properties of the system and for the qualitative evaluation of the kinetic processes and their possible influence on technological devices

  12. Reduction of sidewall interface recombination in GaAs and InGaAs active regions

    Strand, Timothy Andrew

    In the continual effort to reduce the operating current in semiconductor lasers, the first step is always to reduce the size of the device. When we do so, however, we encounter a new set of challenges. As the device size decreases, the "walls close in" on the electrons and holes, that is, the sidewalls of the device become so close together that the electrons and holes can diffuse to them before recombining radiatively. The device sidewalls, are often littered with carrier traps, which act as nonradiative recombination sites for the electrons and holes. This wasted current, a small fraction of the total in larger devices, becomes the dominant current mechanism in small devices. In this work we present two techniques for limiting this sidewall interface recombination. The first uses semiconductor regrowth to remove the recombination sites that are normally formed at the air-exposed sidewalls. We use buried, in-plane lasers to demonstrate a reduction in the sidewall recombination rate by a factor of forty. In the second technique, we show that the sidewall interface recombination can also be reduced by preventing the carriers from diffusing to the sidewalls. We demonstrate two methods for reducing this lateral carrier diffusion; segmented GaAs quantum wells, and InGaAs quantum dots. In the former, we demonstrate a reduction in the low-temperature lateral carrier diffusion constant by a factor of forty-six (versus a comparable GaAs quantum well).

  13. Acoustic emission during hydrogen absorption and desorption in palladium

    Acoustic emission technique has been used to study charging and discharging of hydrogen in palladium. During charging, breaking of oxide film due to surface activation and saturation of hydrogen absorption have been identified by acoustic emission. In the discharging cycle, the desorption of hydrogen from the specimen leads to high AE activity immediately after initiation of discharging, followed by gradual decrease in the acoustic activity, which reaches a minimum upon completion of the desorption. The potential of the acoustic emission technique for studying the kinetics of hydrogen absorption and desorption in metals has been shown. (author)

  14. Enhancement of antibody class-switch recombination by the cumulative activity of four separate elements.

    Dunnick, Wesley A; Shi, Jian; Zerbato, Jennifer M; Fontaine, Clinton A; Collins, John T

    2011-11-01

    Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3' of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3' enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic μ expression is reduced by deletion of the four 3' enhancers. We also test deletions of two or three of the 3' enhancers and show that deletion of more 3' enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3' enhancers; different H chain genes are affected by different 3' enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements. PMID:21949022

  15. In vivo kinetics of [sup 99m]Tc labeled recombinant tissue plasminogen activator in rabbits

    Itoh, Kazuo; Ieko, Masahiro; Hiraguchi, Etsuro; Kitayama, Hide; Tsukamoto, Eriko (Hokkaido Univ., Sapporo (Japan). School of Medicine)

    1994-08-01

    Our previous studies demonstrated that [sup 99m]Tc labeled recombinant tissue plasminiogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of [sup 99m]Tc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr.7) of larger molecular weight than that (Fr.9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: (1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or (2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA. (author).

  16. Characterization of a defensin from the oyster Crassostrea gigas - Recombinant production, folding, solution structure, antimicrobial activities, and gene expression

    Gueguen, Yannick; Herpin, Amaury; Aumelas, André; Garnier, Julien; Fievet, Julie; Escoubas, Jean-Michel; Bulet, Philippe; Gonzalez, Marcelo; Lelong, Christophe; Favrel, Pascal; Bachere, Evelyne

    2006-01-01

    In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spec...

  17. Intravenous Recombinant Tissue Plasminogen Activator Thrombolysis in a Patient with Acute Ischemic Stroke Secondary to Aortic Dissection

    Hong, Keun-Sik; Park, So-Young; Whang, Seon-Il; Seo, So-Young; Lee, Dong-Ha; Kim, Han-Joon; Cho, Joong-Yang; Cho, Yong-Jin; Jang, Woo-Ik; Kim, Chang Young

    2008-01-01

    Background Acute ischemic stroke secondary to aortic dissection (AoD) is challenging in the era of thrombolysis owing to the diagnostic difficulty within a narrow time window and the high risk of complications. Case Report A 64-year-old woman with middle cerebral artery occlusion syndrome admitted to the emergency room within intravenous recombinant tissue plasminogen activator (rt-PA) time window. Her neurological symptoms improved during thrombolysis, but chest and abdominal pain developed....

  18. Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase

    Hu, Wenjun; Begum, Nasim A.; Mondal, Samiran; Stanlie, Andre; Honjo, Tasuku

    2015-01-01

    The B-cell–specific antibody gene-diversifying enzyme activation-induced cytidine deaminase (AID) shows high homology with apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC1), which edits apolipoprotein B-100 mRNA in the presence of cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Here we show that the DNA cleavage and recombination functions of AID depend critically on its RNA-dependent interaction with distinct heterogeneous nuclear ribonu...

  19. Recombinant Mouse PAP Has pH-Dependent Ectonucleotidase Activity and Acts through A1-Adenosine Receptors to Mediate Antinociception

    Sowa, Nathaniel A.; Kunjumon I. Vadakkan; Zylka, Mark J.

    2009-01-01

    Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A1-adenosine receptor (A1R) activation. In this study, we purified the secretory isoform of mouse (m)PAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mP...

  20. Intraocular Lens Opacification following Intracameral Injection of Recombinant Tissue Plasminogen Activator to Treat Inflammatory Membranes after Cataract Surgery

    2015-01-01

    Purpose. To report 7 cases of intraocular lens (IOL) opacification following treatment of postoperative anterior chamber fibrin with recombinant tissue plasminogen activator (rtPA) after cataract surgery. Methods. Retrospective case series of 7 eyes in 7 patients who developed IOL opacification after receiving rtPA for anterior chamber inflammatory membrane formation resulting from phacoemulsification cataract surgery. Three explanted IOLs were investigated with light microscopy, histochemica...

  1. Recombinant human activated protein C inhibits local and systemic activation of coagulation without influencing inflammation during Pseudomonas aeruginosa pneumonia in rats

    Choi, Goda; Hofstra, Jorrit-Jan H; Roelofs, Joris J T H; Florquin, Sandrine; Bresser, Paul; Levi, Marcel; van der Poll, Tom; Schultz, Marcus J

    2007-01-01

    OBJECTIVE: Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that recombinant human activated protein C exerts lung-protective effects via anticoagulant and anti-inflammatory pathways. We investigated the role of the protein C system in pneumonia caused by Pseudomonas aerug

  2. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast.

    Liu, Bo; Shi, DanYang; Chang, ShaoHong; Gong, Xin; Yu, YunZhou; Sun, ZhiWei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens. PMID:25567004

  3. Influence of Different Types of Recombination Active Defects on the Integral Electrical Properties of Multicrystalline Silicon Solar Cells

    Dominik Lausch

    2015-01-01

    Full Text Available In this contribution the influence of different types of recombination-active defects on the integral electrical properties of multicrystalline Si solar cells is investigated. Based on a previous classification scheme related to the luminescence behavior of crystal defects, Type-A and Type-B defects are locally distinguished. It is shown that Type-A defects, correlated to iron contaminations, are dominating the efficiency by more than 20% relative through their impact on the short circuit current ISC and open circuit voltage VOC in standard Si material (only limited by recombination active crystal defects. Contrarily, Type-B defects show low influence on the efficiency of 3% relative. The impact of the detrimental Type-A defects on the electrical parameters is studied as a function of the block height. A clear correlation between the area fraction of Type-A defects and both the global Isc and the prebreakdown behavior (reverse current in voltage regime-2 (−11 V is observed. An outlier having an increased full-area recombination activity is traced back to dense inter- and intragrain nucleation of Fe precipitates. Based on these results it is concluded that Type-A defects are the most detrimental defects in Si solar cells (having efficiencies > 15% and have to be prevented by optimized Si material quality and solar cell process conditions.

  4. An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

    Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P

    2013-01-01

    Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation. PMID:23054694

  5. Evaluation of recombinant activated protein C for severe sepsis at a tertiary academic medical center

    Anger KE

    2013-06-01

    Full Text Available Kevin E Anger,1 Jeremy R DeGrado,1 Bonnie C Greenwood,1 Steven A Cohen,2 Paul M Szumita1 1Department of Pharmacy, Brigham and Women’s Hospital, Boston, MA, USA; 2Department of Family Medicine and Population Health, Division of Epidemiology, Virginia Commonwealth University, Richmond, VA, USA Purpose: Early clinical trials of recombinant human activated protein C (rhAPC for severe sepsis excluded patients at high risk of bleeding. Recent literature suggests bleeding rates are higher in clinical practice and may be associated with worsened outcomes. Our objective was to evaluate baseline demographics; incidence, and risk factors for major bleeding; and mortality of patients receiving rhAPC for severe sepsis at our institution. Methods: A retrospective study was performed for all patients receiving rhAPC for treatment of severe sepsis at a tertiary academic medical center from January 2002 to June 2009. Demographic information, clinical variables, intensive care unit, and hospital outcomes were recorded. Results: Of the 156 patients that received rhAPC, 54 (34.6% did not meet institutional criteria for safe use at baseline due to bleeding precaution or contraindication. Twenty-three (14.7% patients experienced a major bleeding event. Multivariate analysis demonstrated baseline International Normalized Ratio ≥2.5 (odds ratio [OR] 3.68, 95% confidence interval [CI]: 1.28–10.56; P = 0.03 and platelet count ≤100 × 103/mm3 (OR 2.86, 95% CI: 1.07–7.67; P = 0.01 as significant predictors of a major bleed. Overall hospital mortality was 57.7%. Multivariate analysis demonstrated the presence of ≥3 organ dysfunctions (OR 2.46, 95% CI: 1.19–5.09; P < 0.05 and medical intensive care unit admission (OR 1.99, 95% CI: 1.00–3.98; P = 0.05 were independent variables associated with hospital mortality. Conclusion: Patients receiving rhAPC at our institution had higher APACHE II scores, mortality, and major bleeding events than published

  6. β-苯乙醇在不同类型多孔材料上的脱附活化能%Desorption activation energy ofβ-phenylethyl alcohol on different type of porous materials

    王明锋; 熊智勇; 刘娟; 黄艳

    2014-01-01

    采用程序升温脱附(TPD)实验技术测定了β-苯乙醇在5种多孔材料上的TPD曲线,并估算了它们的脱附活化能。同时还讨论了多孔材料的孔径结构和表面性质对β-苯乙醇吸附结合力的影响规律。结果表明:(1)β-苯乙醇在多孔材料上的脱附活化能(kJ/mol)由低到高依次为:B型硅胶(46.90)、SBA-15分子筛(56.16)、A型硅胶(67.92)、MCM-41分子筛(86.16)和XF型活性炭(91.87);(2)除XF型活性炭外,吸附剂表面酸性基团的增加有利于增强其对β-苯乙醇的吸附结合力,β-苯乙醇在吸附剂上的脱附活化能随吸附剂表面酸性基团含量的升高而增大;(3)与其它几种多孔材料相比,XF型活性炭与β-苯乙醇之间因存在π键吸附使得吸附作用力增强,脱附活化能也较高。%Temperature programmed desorption (TPD) technique was applied to measure TPD curves ofβ-phenethyl alcohol on five types of porous materials, and desorption activation energy of phenethyl alcohol was estimated. Effects of pore structure and surface properties of these porous materials on desorption activation energy of phenyl ethyl alcohol were also discussed. Results showed that:(1) Desorption activation energy (kJ/mol) ofβ-phenethyl alcohol on porous materials was in the following order:silica gel B(46.90 kJ/mol)activated carbon XF (91.87 kJ/mol);(2) With exception of activated carbon XF, the higher the surface acidic group content of an adsorbent, the stronger the interaction ofβ-phenethyl alcohol that adsorbent became, and the higher the desorption activation energy;(3) Since there was strongπ-Complexation betweenβ-phenylethyl alcohol and activated carbon, desorption activation energy ofβ-phenethyl alcohol on activated carbon was higher compared to that on other porous materials.

  7. Direct determination of naturally occurring biologically active compound–serum albumin conjugate by matrix-assisted laser desorption/ionization mass spectrometry

    Tanaka, H.; Xuan, L.J.; S. Morimoto; Shoyama, Y.; Isobe, R.; Nojima, K.

    2001-01-01

    Opium alkaloids were conjugated with bovine serum albumin (BSA) to give individual antigen conjugates which were analyzed using BSA as an internal standard by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It became clear that 9 molecules of thebaine were conjugated in a thebaine–BSA conjugate. Codeine and morphine contents in individual conjugates were determined to be 12 and 6 molecules, respectively. Using a relative molecular mass of BSA, 11.5 molecules of forskoli...

  8. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  9. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

    Ayako Chino

    Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

  10. Isolation and characterization of recombinant DNAs containing repeated elements of barley genome: identification of individual actively transcribed families of repeats

    A bank of Escherichia coli clones containing fragments of barley nuclear DNA was obtained using plasmid pBR 322. Clones carrying repeated sequences of the plant genome were selected by means of colony and blot hybridization. Clones with actively transcribed sequences were selected by hybridization to complementary DNA synthesized by means of reverse transcription on a template of total barley poly(A)-containing RNA. Individual families of repeats, two of which contained transcriptionally active sequences of the barley genome, were identified by blot hybridization of recombinant plasmids containing labeled DNA fragments of the inserts of three different clones

  11. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na6[TeW6O24] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  12. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  13. Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

    de Jonge, Evert; Friederich, Philip W.; Vlasuk, George P.; Rote, William E.; Vroom, Margaretha B.; Levi, Marcel; van der Poll, Tom

    2003-01-01

    The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa. PMID:12738659

  14. Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

    de Jonge, Evert; Friederich, Philip W.; Vlasuk, George P.; Rote, William E.; Vroom, Margaretha B.; Levi, Marcel; van der Poll, Tom

    2003-01-01

    The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue f...

  15. Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

    Buhrman, Jason S; Cook, Laura C.; Rayahin, Jamie E; Federle, Michael J.; Gemeinhart, Richard A.

    2013-01-01

    Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. ...

  16. Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

    Alexandra Castilho; Laura Neumann; Pia Gattinger; Richard Strasser; Karola Vorauer-Uhl; Thomas Sterovsky; Friedrich Altmann; Herta Steinkellner

    2013-01-01

    Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomp...

  17. Optimisation of recombinant production of active human cardiac SERCA2a ATPase

    Antaloae, Ana V.; Cédric Montigny; Marc le Maire; Watson, Kimberly A.; Thomas L-M Sørensen

    2013-01-01

    Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain...

  18. Inhibition on the production of collagen type Ⅰ, Ⅲ of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid

    Wen-Bin Liu; Chang-Qing Yang; Wei Jiang; Yi-Qing Wang; Jing-Sheng Guo; Bo-Ming He; Ji-Yao Wang

    2003-01-01

    AIM: To investigate the inhibition effects on the productionof collagen type I, Ⅲ secreted by activated rat hepatic stellatecells (rHSCs) by antisense tissue inhibitors of metalloproteinase1 (TIMP-1) recombinant plasmid through elevating interstitialcollagenase activity.METHODS: rHSCs were extracted from normal rat liverby pronase and collagenase digestion and purified bycentrifugal elutriation, and were cultured on plastic dishesuntil they were activated to a myofibroblastic phenotypeafter 7-10 days. RT-Nest-PCR and gene recombinanttechniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryoticcells. The recombinant plasmid and the pcDNA3 emptyplasmid were transfected in rHSCs by Effectene (QIAGEN)separately. Cells were selected after growing in DMEMcontaining 400 μg/ml G418 for 2-3 weeks. Expression ofexogenous gene was assessed by Northern blot, andexpression oflIMP-1 in rHSCs was determined by Northernblot and Western blot. We tested the interstitial collagenaseactivity with FITC-labled type I collagen as substrate.Ultimately, we quantified the type Ⅰ, Ⅲ collagen byWestern blot.RESULTS: The exogenous antisense TIMP-1 recombinantplasmid could be expressed in rHSCs well, which couldblock the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNAlevel (P<0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98and 1.32 separately at protein level (P<0.05); It mightelevate active and latent interstitial collagenase activity,the collagenase activity was 0.3049, 0.1411 and 0.1196respectively. (P<0.05), which led to promotion thedegradation of type Ⅰ, Ⅲ collagen, the ratio of collagen I/β-actin was 0.63, 1.78 and 1.92 separately (P<0.05); andthe ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98separately (P<0.05).CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on theproduction of type Ⅰ, Ⅲ collagens

  19. Temperature programmed desorption of weakly bound adsorbates on Au(111)

    Engelhart, Daniel P.; Wagner, Roman J. V.; Meling, Artur; Wodtke, Alec M.; Schäfer, Tim

    2016-08-01

    We have performed temperature programmed desorption (TPD) experiments to analyze the desorption kinetics of Ar, Kr, Xe, C2H2, SF6, N2, NO and CO on Au(111). We report desorption activation energies (Edes), which are an excellent proxy for the binding energies. The derived binding energies scale with the polarizability of the molecules, consistent with the conclusion that the surface-adsorbate bonds arise due to dispersion forces. The reported results serve as a benchmark for theories of dispersion force interactions of molecules at metal surfaces.

  20. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  1. Characterisation of aroma profiles of commercial sufus by odour activity value, gas chromatography-olfactometry, aroma recombination and omission studies.

    Xiao, Zuobing; Shang, Yi; Chen, Feng; Niu, Yunwei; Gu, Yongbo; Liu, Shengjiang; Zhu, Jiancai

    2015-01-01

    Sufu is a solid-state fermented product made from soya beans. For the sake of quality control and regulation purposes, it is essential to be able to identify key odorants of various commercial sufus. To identify the aroma-active compounds in sufus, gas chromatography-olfactometry/aroma extract dilution analysis (GC-O/AEDA) was performed, and odour activity value (OAV) was estimated. The correlations between aroma profiles and identified aroma-active compounds were also investigated by principal component analysis. Results showed that 35 aroma-active compounds were detected through OAV calculation, while 28 compounds were identified by using GC-O/AEDA. Quantitative descriptive analysis revealed that aroma recombination model based on OAV calculation was more similar to original sufu in terms of aroma comparing to aroma recombination model based on GC-O/AEDA. Omission experiments further confirmed that the aroma compounds, such as ethyl butanoate, ethyl 2-methylbutanoate, ethyl hexanoate, (E,E)-2,4-decadienal and 2,6-dimethylpyrazine, contributed most significantly to the characteristic aroma of a commercial sufu. PMID:25790084

  2. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

  3. Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

    Suma, Yanasinee; Lim, Heejun; Kwean, Oh Sung; Cho, Suyeon; Yang, Junwon; Kim, Yohan; Kang, Christina S; Kim, Han S

    2016-06-01

    This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use. PMID:26810145

  4. Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

    Gao-Feng Qiu

    2009-01-01

    Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

  5. Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

    Huan, Jianya Y; Meza-Romero, Roberto; Mooney, Jeffery L.; Yuan K. Chou; Edwards, David M.; Rich, Cathleen; Link, Jason M.; Vandenbark, Arthur A.; Bourdette, Dennis N.; Bächinger, Hans-Peter; Burrows, Gregory G.

    2004-01-01

    Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-de...

  6. Recombinant Bactericidal/Permeability-Increasing Protein (rBPI21) in Combination with Sulfadiazine Is Active against Toxoplasma gondii

    Khan, Anis A.; Lambert, Lewis H.; Remington, Jack S.; Araujo, Fausto G.

    1999-01-01

    The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21 for T. gondii was 2.6 μg/ml, whereas only slight cytotoxicity for HFF cells was obse...

  7. Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated, p38 Kinase Spc1 of Fission Yeast

    Gao, Jun; Davidson, Mari K.; Wahls, Wayne P.

    2009-01-01

    Background Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26. Methodology/Principal Findings We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination. Conclusions/Significance The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1. PMID:19436749

  8. A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

    Dong-Ming Lan

    2011-06-01

    Full Text Available A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH42SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short- and medium-chain length p-nitrophenyl (C4 and C8 acyl group esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.

  9. Human Recombinant PLD2 Can Repress p65 Activity of Guinea Pigs of Chronic Asthma in vivo

    Ling Zhu; Weibin Zou; Chuanxing Yu; Junjin Lin; Xiaoli He

    2006-01-01

    This article is to investigate the effect of human recombinant phospholipase D2 (rhPLD2) in vivo on the expression of nuclear transcription factor p65 in chronic asthma of guinea pigs. After treating the guinea pigs with chronic asthma by rhPLD2, the crude nuclear extraction was assayed with TransAM Transcription Factor Assay Kit for the activity of pulmo tissue nuclear transcription factor p65. Compared with the healthy guinea pigs, the activity of nuclear transcription factor p65 in guinea pigs of chronic asthma is much higher than that of control groups. Our results showed that rhPLD2 markedly depressed the activity of p65 when the guinea pigs were attacked by chronic asthma. Cellular & Molecular Immunology. 2006;3(4):307-310.

  10. Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

    Pleckaityte Milda

    2011-11-01

    Full Text Available Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in

  11. Sorption/desorption reversibility of polycyclic aromatic hydrocarbons (PAHs) in soils and carbonaceous materials

    Wang, Guohui

    2008-07-01

    Understanding sorption/desorption is an important prerequisite for the prediction of fate and transport of pollutants in the environment. During the last two decades, numerous studies have reported hysteresis phenomenon for the interaction of hydrophobic organic contaminants (HOCs) with natural organic matter (NOM). It manifests as nonsingular sorption/desorption isotherms or different rates for sorption and desorption, where during desorption a higher affinity of a compound on a given sorbent and a longer time scale for release than for sorption is observed. Other studies showed that some of the reported sorption/desorption hysteresis phenomena are due to experimental artifacts, mainly resulting from non-attainment of sorption equilibrium before desorption experiments, which result in 'pseudo-hysteresis'. Except for the hypothesis of sorbent reconfiguration, clear experimental evidence for the physical or chemical mechanisms proposed to lead to hysteresis is still lacking. In this study, sorption/desorption equilibrium and kinetics of phenanthrene sorption/desorption from two soils and three carbonaceous samples were investigated using both batch and column techniques. The main objective of this work was to monitor hysteresis phenomenon by carefully recovering the solute mass in the system and to compare sorption/desorption equilibria and kinetics thermodynamically. Nonsingular isotherms and higher desorption enthalpies as well as increased activation energies with proceeding desorption are expected if significant hysteresis exists. Sorption-desorption cycles were carried out to compare equilibrium isotherms and associated sorption/desorption enthalpies (AeH, isosteric heats). Instead of the traditional decant-and-refill batch method, the experiments were conducted using a newly designed batch protocol, which enables the determination of sorption/desorption isotherms at different temperatures using a closed batch system. This method additionally allows

  12. Observations of the interaction of a plasma stream with neutral gas: evidence of plasma loss through molecular-activated recombination

    We describe an experiment, the UMIST Linear System (ULS), in which a hydrogen plasma stream, guided by a longitudinal magnetic field, is injected through a diaphragm containing an orifice into a separately-pumped target chamber in which the neutral hydrogen pressure can be raised to a maximum of 8 mTorr. The stream is about 6 mm in diameter, has an electron temperature of up to 15 eV and an ion flux of 3 018s-1; it is supersonic with Mach number up to M approx.= 3. We have studied both the passage of the stream through the orifice and the interaction of the supersonic plasma with neutral hydrogen in the target chamber. We find that transmission is incomplete even when the orifice diameter is five times that of the plasma; we attribute this to the presence of ion trajectories which extend well outside the visible plasma and are intercepted by the diaphragm. In the target chamber, the stream does not broaden, but the ion flux decreases approximately exponentially with distance, with a scale length of the order of the mean free path for momentum transfer in ion-neutral collisions, and much less than that expected for other processes, such as charge exchange or electron-ion recombination. Elastic collisions alone cannot decrease the flux, but would lead to a large accumulation of slow ions in thermal equilibrium with the neutral gas, which must be limited by some other loss process: collisional diffusion and electron-ion recombination are too slow, leading to a density approaching 1020m-3. The observed density is of the order of 1018m-3, requiring a process with a rate of 10-100 times faster. Calculated rates for molecular-activated recombination (MAR) of the slow ions are of this order, and the predicted density agrees with our observations to order of magnitude. (author)

  13. Homing of radiolabelled recombinant interleukin-2 activated natural killer cells and their efficacy in adoptive immunotherapy against murine fibrosarcoma

    Anuradha Rai; Ashim K Chakravarty

    2007-12-01

    Natural killer (NK) cells are spontaneously cytotoxic against tumour target cells. Their number was found to be four times more in the spleen of tumour-bearing Swiss albino mice. After activation with recombinant interleukin-2 (rIL-2), NK cells were tested and found to seek out the tumour site when injected intravenously in tumour-bearing mice. Their potential for fighting tumours in vivo was further seen following adoptive transfer of rIL-2 activated NK (A-NK) cells in tumour-bearing mice. After surgical removal of tumour load, adoptive transfer of A-NK cells inhibited tumour recurrence in 92.3% cases, thereby suggesting the use of this protocol for therapeutic purposes to obtain a better outcome.

  14. Cytosines, but not purines, determine recombination activating gene (RAG)-induced breaks on heteroduplex DNA structures: implications for genomic instability.

    Naik, Abani Kanta; Lieber, Michael R; Raghavan, Sathees C

    2010-03-01

    The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. PMID:20051517

  15. Dissolution of emboli in rats with experimental cerebral thromboembolism by recombinant human tissue plasminogen activator (TD-2061)

    Tissue plasminogen activator (t-PA) is frequently administered clinically as thrombolytic therapy. We injected recombinant t-PA into rats with cerebral 125I-labeled blood clot emboli to evaluate the dissolutive effect of recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA treatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism

  16. Recombinant Expression of a Novel Fungal Immunomodulatory Protein with Human Tumor Cell Antiproliferative Activity from Nectria haematococca

    Shuying Li

    2014-09-01

    Full Text Available To our best knowledge, all of the fungal immunomodulatory proteins (FIPs have been successfully extracted and identified in Basidomycetes, with only the exception of FIP from ascomycete Nectria haematococca (FIP-nha discovered through homology alignment most recently. In this work, a gene encoding FIP-nha was synthesized and recombinantly expressed in an Escherichia coli expression system. SDS-PAGE and MALDI-MS analyses of recombinant FIP-nha (rFIP-nha indicated that the gene was successfully expressed. The yield of the bioactive FIP-nha protein was 42.7 mg/L. In vitro assays of biological activity indicated that the rFIP-nha caused hemagglutination of human and rabbit red blood cells, significantly stimulated mouse spleen lymphocyte proliferation, and enhanced expression of interleukin-2 (IL-2 released from mouse splenocytes, revealing a strong antitumor effect against HL60, HepG2 and MGC823. Through this work, we constructed a rapid and efficient method of FIP production, and suggested that FIP-nha is a valuable candidate for use in future medical care and pharmaceutical products.

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

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

    2012-03-16

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

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

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

  19. Application of synchrotron-radiation-based x-ray microprobe techniques for the analysis of recombination activity of metals precipitated at Si/SiGe misfit dislocations

    In this study we report application of synchrotron-radiation-based x-ray microprobe techniques (the x-ray-beam-induced current (XBIC) and x-ray fluorescence (μ-XRF) methods) to the analysis of the recombination activity and space distribution of copper and iron in the vicinity of dislocations in silicon/silicon-germanium structures. A combination of these two techniques enables one to study the chemical nature of the defects and impurities and their recombination activity in situ and to map metal clusters with a micron-scale resolution. XRF analysis revealed that copper formed clearly distinguishable precipitates along the misfit dislocations. A proportional dependence between the XBIC contrast and the number of copper atoms in the precipitates was established. In hydrogen-passivated iron-contaminated samples we observed clusters of iron precipitates which had no recombination activity detectable by the XBIC technique as well as iron clusters which were not completely passivated

  20. Anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

    The authors showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, they analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation

  1. Thyroid dysfunction induced by recombinant interferon-alpha therapy for chronic active type C hepatitis

    The aim of this study was to assess the frequency and types of thyroid therapy in patients of Chronic dysfunction that develops during IFN- Hepatitis C. The study was carried out on a total of 50 patients of chronic therapy. In addition 50 patients with hepatitis C on recombinant IFN- chronic hepatitis C, not on any antiviral treatment, were included as controls. After informed consent, clinical history was obtained, physical examination was done and findings recorded on a pre-designed proforma. Blood sampling was done for thyroid profile at the beginning of interferon therapy, at 12 weeks and finally at 24 weeks. Thyroid dysfunction (TD) was observed in 14% (n=7) of the patients on antiviral therapy for CHC (n=50). Amongst these seven patients with TD, hypothyroidism was observed in 5 and hyperthyroidism in 2 patients. In contrast the frequency of thyroid dysfunction observed in control group (n=50) was 2%. The frequency of thyroid dysfunction in patients of chronic hepatitis C treated with interferon approaches 14%, with hypothyroidism being the more commonly observed pattern. (author)

  2. Monomer Adsorption-Desorption Processes

    KE Jian-Hong; LIN Zhen-Quan; CHEN Xiao-Shuang

    2009-01-01

    We propose an adsorption-desorption model for a deposit growth system, in which the adsorption and desorption of particles coexist. By means of the generalized rate equation we investigate the cluster (island) size distribution in the dynamic equilibrium state. The results show that the evolution behaviour of the system depends crucially on the details of the rate kernels. The cluster size distribution can take the ecale-frse power-law form in some cases, while it grows exponentially with size in other cases.

  3. Data compilation for particle impact desorption

    The desorption of gases from solid surfaces by incident electrons, ions and photons is one of the important processes of hydrogen recycling in the controlled thermonuclear reactors. We have surveyed the literature concerning the particle impact desorption published through 1983 and compiled the data on the desorption cross sections and desorption yields with the aid of a computer. This report presents the results obtained for electron stimulated desorption, the desorption cross sections and yields being given in graphs and tables as functions of incident electron energy, surface temperature and gas exposure. (author)

  4. Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

    LI Xiao-yan; SHI Wei-wei; WANG Hao; LI Bo-hua; YANG Yang; TAN Min; XUE Jing-ya; GUO Ya-jun

    2005-01-01

    Objective: To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results:rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

  5. Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

    Hansen, Debra T; Thiyagarajan, Thirumagal; Larson, Amy C; Hansen, Jeffrey L

    2016-07-01

    Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting. PMID:26965413

  6. Activity in mice of recombinant BCG-EgG1Y162 vaccine for Echinococcus granulosus infection.

    Ma, Xiumin; Zhao, Hui; Zhang, Fengbo; Zhu, Yuejie; Peng, Shanshan; Ma, Haimei; Cao, Chunbao; Xin, Yan; Yimiti, Delixiati; Wen, Hao; Ding, Jianbing

    2016-01-01

    Cystic hydatid disease is a zoonotic parasitic disease caused by Echinococcus granulosus which is distributed worldwide. The disease is difficult to treat with surgery removal is the only cure treatment. In the high endemic areas, vaccination of humans is believed a way to protect communities from the disease. In this study we vaccinated BALB/c mice with rBCG-EgG1Y162, and then detected the level of IgG and IgE specifically against the recombinant protein by ELISA, rBCG-EgG1Y162 induced strong and specific cellular and humoral immune responses. In vitro study showed that rBCG-EgG1Y162 vaccine not only promote splenocytes proliferation but also active T cell. In addition, the rBCG-EgG1Y162 induced a protection in the mice against secondary infection of Echinococcus granulosus. PMID:26266551

  7. Efficacy of recombinant tissue-type plasminogen activator thrombolysis and primary coronary stenting after acute myocardial infarction

    陈步星; 王伟民; 赵红; 胡大一; 徐成斌; 赵明中; 卢明瑜; 刘健; 吴淳

    2003-01-01

    Objective To compare the efficacy of low dose recombinant tissue-type plasminogen activator (rt-PA) thrombolysis with primary coronary stenting after acute myocardial infarction.Methods Of 261 patients with first acute myocardial infarction, 131 were given low dose rt-PA intravenous thrombolysis, and 130 primary coronary stenting.Results The age, time from onset of chest pain to hospital presentation and infarct location between these two groups were comparable. The patency rate of the infarct-related artery (IRA) in patients in the thrombolysis group was significantly lower than that of patients in the primary stenting group (P0.05).Conclusion Comparing with low dose rt-PA thrombolytic therapy after acute myocardial infarction, primary coronary stenting has a higher patency rate of the IRA, better cardiac function and shorter hospitalization time.

  8. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

    Jeppsson, M.; Johansson, B.; Jensen, Peter Ruhdal;

    2003-01-01

    Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different...... consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic...... transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate...

  9. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [3H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection

  10. Cultured mast cells from asthmatic patients and controls respond with similar sensitivity to recombinant Der P2 induced, IgE-mediated activation

    Krohn, Inge Jacoba Maria Kortekaas; Sverrild, Asger; Lund, Gitte;

    2013-01-01

    conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for...... mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum...

  11. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; Elisabeth S R van den Dungen; Mario Thevis

    2016-01-01

    textabstractAims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human ...

  12. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-09-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

  13. Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

    LI Xiaoyan; WANG Hao; YANG Yang; TAN Min; XUE Jingya; NI Haidong; GUO Yajun

    2006-01-01

    Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

  14. Study of defects near molybdenum surface using thermal desorption spectrometer

    Thermal desorption spectrometry is utilized to study the migration of atoms and defects near molybdenum surface. The thermal desorption spectra of inert gas ions (neon, argon and krypton) injected with various energies (430-1950 eV) into a polycrystalline molybdenum target with various dosages (6.4 x 10sup(12) - 3.9 x 10sup(14) ions/cmsup(2)) are investigated. Four different states of binding of the trapped atoms corresponding to the activation energies for desorption have been revealed from the spectra. The activation energies are found to be relatively insensitive to the species of the bombarding ion, incident ion energy and the dosage. The patterns of the spectra are strongly influenced by the mean projected range of the ions into the solid. The activation energies deduced are in good agreement with those reported for the migration of atoms and defects in molybdenum. (auth.)

  15. SPS Ion Induced Desorption Experiment

    Maximilien Brice

    2003-01-01

    This experiment will give a study about the induced desorption from heavy ion (Indium ion run from week 45 in SPS T4-H8 area) impacting LHC type graphite collimator. 4 different samples are located in the 4 chambers 90° one to each other: pure graphite, graphite with copper coating, graphite with NEG coating, 316LN stainless steal (reference).

  16. Adjuvant Effects for Oral Immunization Provided by Recombinant Lactobacillus casei Secreting Biologically Active Murine Interleukin-1β▿

    KAJIKAWA, Akinobu; MASUDA, Kazuya; Katoh, Mitsunori; Igimi, Shizunobu

    2009-01-01

    Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1β (IL-1β), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1β as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, inc...

  17. 78 FR 66751 - Office of Science Policy, Office of Biotechnology Activities; Recombinant or Synthetic Nucleic...

    2013-11-06

    ... HUMAN SERVICES National Institutes of Health Office of Science Policy, Office of Biotechnology... Biotechnology Activities (OBA) is updating Appendix B (Classification of Human Etiologic Agents on the Basis of... mail to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge...

  18. Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide.

    Rothan, Hussin A; Mohamed, Zulqarnain; Suhaeb, Abdulrazzaq M; Rahman, Noorsaadah Abd; Yusof, Rohana

    2013-11-01

    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection. PMID:24044366

  19. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- XTM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-XTM genome, which was transformed into E. coli to get recombinant Adeno-XTM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 1011 TCID50/ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-XTM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

  20. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  1. A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells.

    Nalivaiko, Kristina; Hofmann, Martin; Kober, Karina; Teichweyde, Nadine; Krammer, Peter H; Rammensee, Hans-Georg; Grosse-Hovest, Ludger; Jung, Gundram

    2016-02-01

    Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease. PMID:26581163

  2. Recombinant human interleukin 5 is a selective activator of human eosinophil function

    1988-01-01

    Human rIL-5 was found to selectively stimulate morphological changes and the function of human eosinophils. This molecule is thus a prime candidate for the selective eosinophilia and eosinophil activation seen in disease.

  3. Anti-invasive and anti-adhesive activities of a recombinant disintegrin, r-viridistatin 2, derived from the Prairie rattlesnake (Crotalus viridis viridis)

    Lucena, Sara E; Jia, Ying; Soto, Julio G.; Parral, Jessica; Cantu, Esteban; Brannon, Jeremy; Lardner, Kristina; Ramos, Carla J.; Seoane, Agustin I.; Sánchez, Elda E

    2012-01-01

    Snake venom disintegrins inhibit platelet aggregation and have anti-cancer activities. In this study, we report the cloning, expression, and functional activities of a recombinant disintegrin, r-viridistatin 2 (GenBank ID: JQ071899), from the Prairie rattlesnake. r-Viridistatin 2 was tested for anti-invasive and anti-adhesive activities against six different cancer cell lines (human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorecta...

  4. Effect of dexamethasone on peroxisome proliferator activated receptor-gamma mRNA expression in 3T3-L1 adipocytes with the human recombinant adiponectin

    SHE Qi-mei; ZHAO Jing; WANG Xia-lian; ZHOU Chang-man; SHI Xian-zhong

    2007-01-01

    Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin (ADPN) function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma (PPAR-γ) mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. Methods The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1 + were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1+ were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1+-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent (Qiagen). Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone (0.5 mmol/L) for 24 hours, cells were collected and total RNA was extracted. The PPAR-γ mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Results After eukaryotic recombinant was digested by Hind Ⅲ and EcoR Ⅰ, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone (0.5 mmol/L) decreased PPAR-γ mRNA expression compared to untreated controls (P<0.01). Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.Conclusion The 3T3-L1 cells stably transfected human recombinant

  5. Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect

    Mathijssen, N.C.J.; Masereeuw, R.; Holme, P.A.; Kraaij, M.G.J. van; Laros, B.A.P.; Peyvandi, F.; Heerde, W.L. van

    2013-01-01

    INTRODUCTION: Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. MATERIALS AND METHODS: Ten factor VII deficient patients

  6. The biological activity of a recombinantly expressed (His)(6)-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal

    Jensen, Louise Bjerremann; Torp, Anna Maria; Andersen, Sven Bode;

    2008-01-01

    The application of recombinant (His)(6)-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it is...

  7. Production of mmLFA3-Ig with immunosuppressive activity by recombinant expression in Pichia pastoris

    Jian Zhu; Shengyun Zhu

    2013-01-01

    Engagement of lymphocyte function antigen 3 (LFA3) and CD2 can increase the activation of T cells. Blockade of LFA3-CD2 pathway efficiently down-regulated the abnormal immune activation, thus resulting in the development of human LFA3-Ig (hLFA3-Ig) as a treatment for psoriasis. Nevertheless, it is far from to unravel the role of LFA3-CD2 in regulation of immune system. Rhesus monkey macaca mulatta has been widely used as animal models for human immune disorders. Due to specificity of the immu...

  8. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

  9. Antiviral activity of recombinant porcine surfactant protein A against porcine reproductive and respiratory syndrome virus in vitro.

    Li, Lan; Zheng, Qisheng; Zhang, Yuanpeng; Li, Pengcheng; Fu, Yanfeng; Hou, Jibo; Xiao, Xilong

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the swine industry worldwide. However, there is not an ideal vaccine to provide complete protection against PRRSV. Thus, the need for new antiviral strategies to control PRRSV still remains. Surfactant protein A (SP-A) belongs to the family of C-type lectins, which can exert antiviral activities. In this present study, we assessed the antiviral properties of recombinant porcine SP-A (RpSP-A) on PRRSV infection in Marc 145 cells and revealed its antiviral mechanism using a plaque assay, real-time qPCR, western blotting analysis and an attachment and penetration assay. Our results showed that RpSP-A could inhibit the infectivity of PRRSV in Marc 145 cells and could reduce the total RNA and protein level. The attachment assay indicated that RpSP-A in the presence of Ca(2+) could largely inhibit Marc 145 cell attachment; however, in the penetration assay, it was relatively inactive. Furthermore, our study suggested that virus progeny released from infected Marc145 cells were blocked by RpSP-A from infecting other cells. We conclude that RpSP-A has antiviral activity against PRRSV, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, RpSP-A holds promise as a novel antiviral agent against PRRSV. PMID:27101074

  10. Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

    Huan, Jianya Y; Meza-Romero, Roberto; Mooney, Jeffery L; Chou, Yuan K; Edwards, David M; Rich, Cathleen; Link, Jason M; Vandenbark, Arthur A; Bourdette, Dennis N; Bächinger, Hans-Peter; Burrows, Gregory G

    2012-01-01

    Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. PMID:22973070

  11. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. PMID:26903242

  12. Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity.

    Huan, Jianya Y; Meza-Romero, Roberto; Mooney, Jeffery L; Chou, Yuan K; Edwards, David M; Rich, Cathleen; Link, Jason M; Vandenbark, Arthur A; Bourdette, Dennis N; Bächinger, Hans-Peter; Burrows, Gregory G

    2005-01-01

    Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. PMID:22973070

  13. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-ZeoR marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53+/- mouse fibroblasts show elevated levels of homologous recombination compared to their p53+/+ counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors

  14. Inhibitory activity of Filipendula ulmaria constituents on recombinant human histidine decarboxylase.

    Nitta, Yoko; Kikuzaki, Hiroe; Azuma, Toshiaki; Ye, Yuan; Sakaue, Motoyoshi; Higuchi, Yoshiki; Komori, Hirohumi; Ueno, Hiroshi

    2013-06-01

    Histidine decarboxylase (HDC) catalyses the formation of histamine, a bioactive amine. Agents that control HDC activity are beneficial for treating histamine-mediated symptoms, such as allergies and stomach ulceration. We searched for inhibitors of HDC from the ethyl acetate extract of the petal of Filipendula ulmaria, also called meadowsweet. Rugosin D, rugosin A, rugosin A methyl ester (a novel compound), and tellimagrandin II were the main components; these 4 ellagitannins exhibited a non-competitive type of inhibition, with K(i) values of approximately 0.35-1 μM. These K(i) values are nearly equal to that of histidine methyl ester (K(i)=0.46 μM), an existing substrate analogue inhibitor. Our results show that food products contain potent HDC inhibitors and that these active food constituents might be useful for designing clinically available HDC inhibitors. PMID:23411280

  15. Efficacy and effectiveness of recombinant human activated protein C in severe sepsis of adults

    Greiner, Wolfgang; Willich, Stefan N.; Roll, Stefanie; Müller-Nordhorn, Jaqueline; Schumacher, Helge Knut

    2007-01-01

    Introduction: Sepsis is defined as an invasion of microorganisms and/or their toxins into the blood associated the reaction of the organism to this invasion. Severe sepsis is a major cost driver in intensive care medicine. In Germany, prevalence data was assessed in the context of the German Prevalence Study. Severe sepsis has a prevalence of 35% in German intensive care units. Research questions: The following questions were analysed: is Drotrecogin alfa (activated) (DAA) effective in the tr...

  16. Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test

    Boldrin, Paula Karina; Resende, Flávia Aparecida; Höhne, Ana Paula Oliveira; de Camargo, Mariana Santoro; Espanha, Lívia Greghi; Nogueira, Catarine Haidê; Melo, Maria do Socorro F; Vilegas, Wagner; Varanda, Eliana Aparecida

    2013-01-01

    Background Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as “rattle or rattlesnake” and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of ...

  17. Sustained thrombolysis with DNA-recombinant tissue type plasminogen activator in rabbits

    Tissue type plasminogen activator (t-PA) is an effective thrombolytic agent in experimental animals. The duration of the thrombolytic effect of infused t-PA is unknown. The authors compared the duration of the thrombolytic effect of t-PA with streptokinase by measuring the lysis of 125I-fibrin-labeled thrombi in rabbit jugular veins at different times after a bolus injection of the fibrinolytic agents. The pharmacodynamics of both thrombolytic agents were determined in rabbits using a sensitive ex vivo fibrinolytic assay. Streptokinase and t-PA were given as a bolus dose of 15,000 U/kg. There was no detectable circulating fibrinolytic activity 30 minutes after the bolus dose of t-PA and 120 minutes after the bolus dose of streptokinase. The t-PA injection produced 34% thrombolysis at 30 minutes, 90% thrombolysis at 120 minutes, and 96% thrombolysis at 240 minutes. The streptokinase injection produced 17% thrombolysis at 30 minutes, 34% at 120 minutes, and 34% at 240 minutes. These observations indicate that the thrombolytic effect of t-PA is sustained beyond its time of clearance from the circulation whereas the thrombolytic effect of streptokinase closely parallels its activity in the circulation

  18. High production in E. coli of biologically active recombinant human fibroblast growth factor 20 and its neuroprotective effects.

    Tian, Haishan; Zhao, Yang; Chen, Nazi; Wu, Meiyu; Gong, Weiyue; Zheng, Jie; Fernig, David G; Jungbauer, Alois; Wang, Dezhong; Li, Xiaokun; Jiang, Chao

    2016-04-01

    Fibroblast growth factor 20 (FGF20) has a wide range of biological activities; its expression is most pronounced in neural tissues where it has functions in development and neuroprotection. Given these activities, interest in the clinical applications of FGF20 is rising, which will lead to increasing demand for active recombinant human FGF20 (rhFGF20). To improve the production of rhFGF20, an artificial gene encoding fgf20 was cloned into pET3a and expressed in E. coli BL21(DE3)pLysS. By optimizing induction conditions, we successfully induced large amounts of insoluble rhFGF20. Following solubilization and refolding of the rhFGF20 from inclusion bodies, it was purified by HiTrap heparin affinity chromatography to a purity of over 96 % with a yield of 218 mg rhFGF20/100 g wet cells. The purified rhFGF20 could stimulate proliferation of both NIH 3T3 cells and PC-12 cells, measured by the MTT assay. In a model of Aβ25-35-induced apoptosis on PC-12 cells, rhFGF20 had a clear protective effect. RT-PCR and Western blot analysis of apoptosis-related genes and proteins revealed that the FGF20-derived protective mechanism was likely due to the relief of endoplasmic reticulum stress (ER stress). In conclusion, the approach described here may be a better means to produce active rhFGF20 in good quantity, thereby allowing for its future pharmacological and clinical use. PMID:26603761

  19. Effect of Ultrasound on Desorption Equilibrium

    秦炜; 原永辉; 戴猷元

    2001-01-01

    Effects of ultrasound on intensification of separation process were investigated through the experiment of desorption equilibrium behavior. Tri-butyl phosphate (TBP) on NKA-X resin and phenol on a solvent impregnated resin, CL-TBP resin, were used for desorption processes. The desorption rate was measured with and without ultrasound. Desorption equilibrium was studied under various ultrasonic power densities or thermal infusion. Results showed that the desorption rate with ultrasound was much higher than that with normal thermal infusion. Both ultrasound and thermal infusion broke the desorption equilibrium existed at room temperature. However, after the systems were cooled down, the amount of solute desorbed in the liquid phase in the presence of ultrasound was much higher than that at the temperature corresponding to the same ultrasound power. It is proved that the initial desorption equilibrium was broken as a result of the spot energy effect of ultrasound.

  20. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  1. Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

    Rebuffat, S A; MORIN, M; Nguyen, B; Castex, F; Robert, B.; Péraldi-Roux, S.

    2010-01-01

    Background: Thyroid cancers are difficult to treat due to their limited responsiveness to chemo- and radiotherapy. There is thus a great interest in and a need for alternative therapeutic approaches. Results: We studied the cytotoxic activity of anti-thyroperoxidase autoantibodies (anti-TPO aAbs, expressed in baculovirus/insect cell (B4) and CHO cells (B4′) or purified from patients' sera) against a papillary thyroid cancer (NPA) cell line. Anti-TPO aAbs from patients' sera led to a partial d...

  2. Myocardial infarction following recombinant tissue plasminogen activator treatment for acute ischemic stroke: a dangerous complication

    ZHOU Zhi-gang; WANG Rui-lan; YU Kang-long

    2012-01-01

    Thrombolysis with intravenous tissue plasminogen activator (t-PA) is currently an approved therapy for patients with acute ischemic stroke.Acute myocardial infarction (AMI) immediately following t-PA treatment for stroke is a rare but serious complication.A case of acute myocardial infarction (MI) following IV t-PA infusion for acute stroke was observed.This is a 52-year-old male with a known history of hypertension and chest pain,who subsequently developed MI four hours after IV t-PA was administered for acute ischemic stroke.The disruption of intra-cardiac thrombus and subsequent embolization to the coronary arteries may be an important mechanism.In addition.spontaneous recanalization of infarct-related arteries may be associated with 9reater myocardial salvage and better prognosis.

  3. Analysis of the Peroxidase Activity of Rice (Oryza Sativa) Recombinant Hemoglobin 1: Implications for the In Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about the peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH ...

  4. T-Independent Activation-Induced Cytidine Deaminase Expression, Class-Switch Recombination, and Antibody Production by Immature/Transitional 1 B Cells1

    Ueda, Yoshihiro; Liao, Dongmei; Yang, Kaiyong; Patel, Anjali; Kelsoe, Garnett

    2007-01-01

    Inflammation elicits a splenic lymphopoiesis of unknown physiologic significance but one that juxtaposes developing B cells and exogenous Ag. We show that immature and transitional 1 (immature/T1) B cells constitutively express activation-induced cytidine deaminase and B lymphocyte-induced maturation protein 1 in amounts that support accelerated plasmacytic differentiation and limited class-switch recombination. In vivo, activation of immature/T1 B cells by TLR ligands or bacterial vaccine ra...

  5. Potentiation of mitomycin C and porfiromycin antitumor activity in solid tumor models by recombinant human interleukin 1 alpha.

    Braunschweiger, P G; Jones, S A; Johnson, C S; Furmanski, P

    1991-10-15

    The time- and dose-dependent effects of recombinant human interleukin 1 alpha (IL-1 alpha) on the antitumor activity of mitomycin C (MMC) and porfiromycin (PORF) were studied in RIF-1 and Panc02 solid tumor model systems. IL-1 alpha produced dose-dependent sensitization of clonogenic RIF-1 tumor cells to MMC in vivo. IL-1 alpha chemosensitization was highly schedule dependent, and the most efficacious schedules produced dose-modifying factors of 3.6 and 5.1 for MMC and PORF, respectively. More than additive clonogenic cell kill after IL-1 alpha-chemotherapy combinations reflected increased cellular sensitivity to MMC and PORF. The combinations also produced marked decreases in the yield of viable tumor cells, suggesting that the bioreductive drugs may have also potentiated the microvascular injury and ischemia produced by IL-1 alpha. Dexamethasone inhibited and ketoconazole, an inhibitor of corticosterone biosynthesis, enhanced IL-1 alpha-mediated chemosensitization in these models. IL-1 alpha mediated chemosensitization to MMC, and PORF was also demonstrated by tumor growth inhibition in the RIF-1 model and increased survival of mice in the spontaneously metastasizing Panc02 system. Chemosensitization of bone marrow spleen colony-forming units was not seen. IL-1 alpha (1000 units/ml) had no effect on MMC and PORF cytotoxicity in RIF-1 and PORF cell lines in vitro. The results indicate that the tumor-specific IL-1 alpha-induced pathophysiologies can sensitize solid tumors to agents which are preferentially activated, retained, and cytotoxic to cells under hypoxic conditions. Our results suggest that strategies combining bioreductively activated hypoxic cell cytotoxins and biological agents might offer efficacious alternatives or adjuvants to conventional combination approaches. PMID:1913664

  6. Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

    Meshaal, Safa; El Hawary, Rabab; Elsharkawy, Marwa; Mousa, Reem K; Farid, Reem J; Abd Elaziz, Dalia; Alkady, Radwa; Galal, Nermeen; Massaad, Michel J; Boutros, Jeannette; Elmarsafy, Aisha

    2015-06-01

    The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID. PMID:25869295

  7. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure. PMID:23716035

  8. ALMA observations of the submillimetre hydrogen recombination line from the type 2 active nucleus of NGC 1068

    Izumi, Takuma; Nakanishi, Kouichiro; Imanishi, Masatoshi; Kohno, Kotaro

    2016-07-01

    Hydrogen recombination lines at the submillimetre band (submm-RLs) can serve as probes of ionized gas without dust extinction. One therefore expects to probe the broad-line region (BLR) of an obscured (type 2) active galactic nucleus (AGN) with those lines. However, admitting the large uncertainty in the continuum level, here we report on the non-detection of both broad and narrow H26 α emission line (rest frequency = 353.62 GHz) towards the prototypical type 2 AGN of NGC 1068 with the Atacama Large Millimeter/submillimeter Array (ALMA). We also investigate the nature of BLR clouds that can potentially emit submm-RLs with model calculations. As a result, we suggest that clouds with an electron density (Ne) of ˜109 cm-3 can mainly contribute to broad submm-RLs in terms of the line flux. On the other hand, line flux from other density clouds would be insignificant considering their too large or too small line optical depths. However, even for the case of Ne ˜ 109 cm-3 clouds, we also suggest that the expected line flux is extremely low, which is impractical to detect even with ALMA.

  9. Hemothorax under thrombolytic therapy with recombinant tissue: plasminogen activator (rt-PA) in a 16-year-old girl.

    Varnholt, V; Ringe, H; Nietsch, L; Gaedicke, G

    1999-12-01

    We present the case of a 16-year-old girl with an extended thrombosis of the femoral and iliac vein and the inferior vena cava during pleuropneumonia; predisposing risk factors for thrombophilia were: use of contraceptives, nicotine abuse and congenital deficiency of antithrombin III (not previously diagnosed). Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA; initial dose: 0.08 mg/kg/h) was started. 2 days later--after diagnosis of an extended hemothorax: 1500 ml blood were obtained after thoracocentesis, transfusion of packed red blood cells was necessary--rt-PA was stopped, with only heparin (400 U/kg/d) being administered. 36 h later--the thrombosis had not yet changed--the thrombolytic therapy with rt-PA was continued in a markedly reduced dose (0.015 mg/kg/d) with no further bleeding complications. 8 days later--after successful thrombolysis--t-PA was stopped, heparin was given for another 10 days, then cumarin was administered orally. PMID:10650854

  10. ALMA observations of the submillimetre hydrogen recombination line from the type 2 active nucleus of NGC 1068

    Izumi, Takuma; Imanishi, Masatoshi; Kohno, Kotaro

    2016-01-01

    Hydrogen recombination lines at the submillimetre band (submm-RLs) can serve as probes of ionized gas without dust extinction. One therefore expects to probe the broad line region (BLR) of an obscured (type 2) active galactic nucleus (AGN) with those lines. However, admitting the large uncertainty in the continuum level, here we report on the non-detection of both broad and narrow H26$\\alpha$ emission line (rest frequency = 353.62 GHz) towards the prototypical type 2 AGN of NGC 1068 with the Atacama Large Millimeter/submillimeter Array (ALMA). We also investigate the nature of BLR clouds that can emit submm-RLs with model calculations. As a result, we suggest that clouds with an electron density ($N_e$) of $\\sim$ 10$^9$ cm$^{-3}$ can mainly contribute to broad submm-RLs in terms of the line flux. On the other hand, line flux from other density clouds would be insignificant considering their too large or too small line optical depths. Since $N_e$ $\\sim$ 10$^9$ cm$^{-3}$ is typical in BLRs, insufficient sensiti...

  11. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Xiaojing Yang

    Full Text Available Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1 in human embryonic kidney 293 (HEK293 cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  12. Recombinant human erythropoietin reduces plasminogen activator inhibitor and ameliorates pro-inflammatory responses following trauma

    M Mojtahedzadeh

    2011-05-01

    Full Text Available "n  "n Background and the purpose of the study: Besides its hematopoietic effects, erythropoietin (EPO by mobilization of iron and modulation of some inflammatory cytokines has antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate these effects of erythropoietin and its impact on organ function in traumatized patients. "n Methods: Twenty-six ICU-admitted traumatized patients within 24 hrs after trauma were randomly assigned to the EPO (received EPO, 300 units/Kg/day and Control (not received EPO groups. The inflammatory biomarkers including Tumor Necrosis Factor alpha (TNF-α, Interleukin 1 (IL-1, Plasminogen Activator Inhibitor 1 (PAI-1 and Nitrotyrosine were recorded at the admission, 3, 6 and 9 days thereafter. Acute Physiology and Chronic Health Evaluation (APACHE II and Sequential Organ Failure Assessment (SOFA scores were also recorded. "n Results: Among 12 patients (EPO group TNF-α level at the day of 9 (P=0.046, and within EPO group at the days of 3 (P=0.026 ameliorate, 6 (P=0.016, and 9 (P=0.052 were significantly lowered. Level of IL-1 and PAI-1 decreased significantly at days of 3, 6 and 9 post intervention. Also there were significant differences between two groups in the SOFA score during three measured time intervals (the first, third and seventh days. "n Conclusion: From the results of this study it seems that injection of erythrocyte stimulating agent is well tolerated and inhibits the inflammatory response and oxidative stress following trauma.

  13. Phase I trial with recombinant interleukin-2 (rIL-2): immune activation by rIL-2 alone or following pretreatment with recombinant interferon-gamma.

    Farace, F; Mathiot, C; Brandely, M; Tursz, T; Dorval, T; Pouillart, P; Triebel, F; Hercend, T; Fridman, W H

    1990-01-01

    Alterations of immunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL-2. Five-day infusions of rIL-2 at doses from 1 x 10(6) to 24 x 10(6) biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 x 10(6) U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analyzed at days 0, 8 and 11 of the rIL-2 courses. Following a leucopenia during rIL-2 infusion, a lymphocytosis was found in all patients except one. The lymphocytosis peaked at day 8 and was detected at doses of rIL-2 as low as 1 x 10(6) U/m2 per day, reaching a plateau at a dose of 16 x 10(6) U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL-2, some patients had predominant T cells (CD3+, NKH1(CD56)-), others had predominant natural killer (NK) cells (CD3-, NKH1 (CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL-2 was preceded by a course of 5 days infusion of 2 x 10(6) U/m2 per day recombinant interferon-gamma (rIFN-gamma). The infusion of rIFN-gamma prior to rIL-2 had no effect on the rIL-2-induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL-2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low-dose rIFN-gamma does not modify the immune stimulation by rIL-2. PMID:2122928

  14. Calculation of activation energies for transport and recombination in mesoporous TiO2/dye/electrolyte films--taking into account surface charge shifts with temperature.

    O'Regan, Brian C; Durrant, James R

    2006-05-01

    Transient photovoltage and photocurrent measurements have been employed to determine the recombination and transport kinetics in operating dye-sensitized photovoltaic cells as a function of potential and temperature. Photocurrent transients have been taken at the open circuit potential, as opposed to the standard measurement at short circuit. Kinetic results have been used to calculate the activation energy as function of the Fermi level position in the TiO(2). In the calculation of activation energies, we have explicitly taken into account the temperature dependence of the offset between the electrolyte redox potential and the conduction band edge. This new method gives activation energies that decrease linearly as the Fermi level position moves toward the conduction band edge, as expected, but not found in previous studies. The results are consistent with the presence of a distribution of traps below the TiO(2) conduction band, the detrapping from which limits both the transport and the recombination of electrons. PMID:16640403

  15. Efficacy and effectiveness of recombinant human activated protein C in severe sepsis of adults

    Greiner, Wolfgang

    2007-07-01

    Full Text Available Introduction: Sepsis is defined as an invasion of microorganisms and/or their toxins into the blood associated the reaction of the organism to this invasion. Severe sepsis is a major cost driver in intensive care medicine. In Germany, prevalence data was assessed in the context of the German Prevalence Study. Severe sepsis has a prevalence of 35% in German intensive care units. Research questions: The following questions were analysed: is Drotrecogin alfa (activated (DAA effective in the treatment of patients with severe sepsis and a mixed risk of death, both in all patients and in different subgroups? Is DAA effective in the treatment of patients with severe sepsis and low risk of death? Is DAA cost effective in the treatment of patients with severe sepsis compared to placebo? Methods: Only studies with adult patients are included. There are no other exclusion criteria. A systematic literature search is performed by the German Institute of Medical Documentation and Information (DIMDI. The literature search yielded as a total of 847 hits. After screening of the abstracts, 165 medical and 101 economic publications were chosen for full text appraisal. Results: Therapy with DAA appears to be cost effective in reducing 28-day-mortality in patients with severe sepsis and a high risk of death. A high risk of death is indicated by the presence of multiorgan failure (≥2 and/or an APACHE-II-Score ≥25. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is cost-effective in patients with multiorgan failure and/or an APACHE II Score (≥25. In patients with a lower risk of death, DAA is not cost-effective. Costs associated with bleeding events have been rarely included in cost calculations. Discussion: DAA appears to reduce mortality in patients with severe sepsis and a high

  16. Decomposition kinetics study of zirconium hydride by interrupted thermal desorption spectroscopy

    Ma, Mingwang; Liang, Li; Tang, Binghua; Xiang, Wei; Wang, Yuan; Cheng, Yanlin; Tan, Xiaohua, E-mail: caepiee@163.com

    2015-10-05

    Highlights: • Interrupted TDS was applied to investigate the mechanism of ZrH{sub 2} decomposition. • The activation energies for the five desorption peaks were determined. • The origins of the five desorption peaks were identified. • The γZrH phase was observed at ambient conditions. - Abstract: Thermal desorption kinetics of zirconium hydride powder were studied using thermogravimetry and simultaneous thermal desorption spectroscopy. The activation energies for observed desorption peaks were estimated according to Kissinger relation. The intermediate phase composition was studied using X-ray diffraction by rapid cooling on different stages of heating. The origins of the peaks were described as the equilibrium hydrogen pressure of a number of consecutive phase regions that decomposition reaction passed through. The zirconium monohydride γZrH was observed for extended periods of time at ambient conditions, which has been supposed to be metastable for a long time.

  17. Effect of recombinant human erythropoietin administration on lipid peroxidation and antioxidant enzyme(s activities in preterm infants.

    Akisu M

    2001-12-01

    Full Text Available In the present investigation, we studied the effect of recombinant human erythropoietin (r-HuEPO on serum malondialdehyde (MDA as an index of lipid peroxidation, related to iron-catalyzed free radical reaction and erythrocyte superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GPX activities in very-low-birth weight (VLBW infants. Forty premature infants, at gestational ages were less than 33 weeks and birthweights were less than 1,500 g, were enrolled in the study. The study population was randomly divided into 2 groups. Twenty infants in Group 1 (treatment group were given r-HuEPO, and 20 infants in Group 2 served as the control. r-HuEPO treatment (750 U/kg a week was initiated on the 10th day of life and continued for 6 weeks. Preterm infants given erythrocyte transfusions during the study were excluded from the results. Serum ferritin and MDA levels, and erythrocyte superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GPX activities were analyzed at the end of the first week of life (at the beginning of the study. Subsequently, serum ferritin, and MDA levels were measured at the end of the 3rd and the 6th week. SOD, CAT, and GPX activities in the hemolysate were analyzed at the end of the 4th week. Six infants in the control group and 1 infant in the r-HuEPO group received transfusions through the end of the study, and these infants were excluded from the results. Significantly decreased serum ferritin concentrations were found in the r-HuEPO group compared to those in the control group both at the end of the 3rd and the 6th week (P < 0.05, and P < 0.01, respectively. In addition, serum MDA levels were also significantly reduced in Group 1 compared to control both at the end of the 3rd and the 6th week (P < 0.01 and P < 0.05, respectively. A good correlation was found between serum MDA and ferritin levels in Group 1. When the 2 groups were compared with respect to activities of SOD, CAT, and GPX at the end

  18. Revisited reaction-diffusion model of thermal desorption spectroscopy experiments on hydrogen retention in material

    Desorption phase of thermal desorption spectroscopy (TDS) experiments performed on tungsten samples exposed to flux of hydrogen isotopes in fusion relevant conditions is analyzed using a reaction-diffusion model describing hydrogen retention in material bulk. Two regimes of hydrogen desorption are identified depending on whether hydrogen trapping rate is faster than hydrogen diffusion rate in material during TDS experiments. In both regimes, a majority of hydrogen released from material defects is immediately outgassed instead of diffusing deeply in material bulk when the evolution of hydrogen concentration in material is quasi-static, which is the case during TDS experiments performed with tungsten samples exposed to flux of hydrogen isotopes in fusion related conditions. In this context, analytical expressions of the hydrogen outgassing flux as a function of the material temperature are obtained with sufficient accuracy to describe main features of thermal desorption spectra (TDSP). These expressions are then used to highlight how characteristic temperatures of TDSP depend on hydrogen retention parameters, such as trap concentration or activation energy of detrapping processes. The use of Arrhenius plots to characterize retention processes is then revisited when hydrogen trapping takes place during TDS experiments. Retention processes are also characterized using the shape of desorption peaks in TDSP, and it is shown that diffusion of hydrogen in material during TDS experiment can induce long desorption tails visible aside desorption peaks at high temperature in TDSP. These desorption tails can be used to estimate activation energy of diffusion of hydrogen in material

  19. Spatially resolved thermal desorption/ionization coupled with mass spectrometry

    Jesse, Stephen; Van Berkel, Gary J; Ovchinnikova, Olga S

    2013-02-26

    A system and method for sub-micron analysis of a chemical composition of a specimen are described. The method includes providing a specimen for evaluation and a thermal desorption probe, thermally desorbing an analyte from a target site of said specimen using the thermally active tip to form a gaseous analyte, ionizing the gaseous analyte to form an ionized analyte, and analyzing a chemical composition of the ionized analyte. The thermally desorbing step can include heating said thermally active tip to above 200.degree. C., and positioning the target site and the thermally active tip such that the heating step forms the gaseous analyte. The thermal desorption probe can include a thermally active tip extending from a cantilever body and an apex of the thermally active tip can have a radius of 250 nm or less.

  20. Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination

    The breast cancer susceptibility protein, Brca2 and the RecQ helicase, Blm (Bloom syndrome mutated) are tumor suppressors that maintain genome integrity, at least in part, through homologous recombination (HR). Brca2 facilitates HR by interacting with Rad51 in multiple regions, the BRC motifs encoded by exon 11 and a single domain encoded by exon 27; however, the exact importance of these regions is not fully understood. Blm also interacts with Rad51 and appears to suppress HR in most circumstances; however, its yeast homologue Sgs1 facilitates HR in response to some genotoxins. To better understand the biological importance of these two proteins, we performed a genotoxic screen on mouse embryonic stem (ES) cells impaired for either Brca2 or Blm to establish their genotoxic profiles (a cellular dose-response to a wide range of agents). This is the first side-by-side comparison of these two proteins in an identical genetic background. We compared cells deleted for Brca2 exon 27 to cells reduced for Blm expression and find that the Brca2- and Blm-impaired cells exhibit genotoxic profiles that reflect opposing activities during HR. Cells deleted for Brca2 exon 27 are hypersensitive to γ-radiation, streptonigrin, mitomycin C and camptothecin and mildly resistant to ICRF-193 which is similar to HR defective cells null for Rad54. By contrast, Blm-impaired cells are hypersensitive to ICRF-193, mildly resistant to camptothecin and mitomycin C and more strongly resistant to hydroxyurea. These divergent profiles support the notion that Brca2 and Blm perform opposing functions during HR in mouse ES cells

  1. Hyperacute thrombolysis with recombinant tissue plasminogen activator of acute ischemic stroke: Feasibility and effectivity from an Indian perspective

    Sharma S

    2008-01-01

    Full Text Available Given the constraints of resources, thrombolysis for acute ischemic stroke (AIS is under evaluation in developing countries like India, especially in areas such as western Utter Pradesh, where it is overly crowded and there is poor affordability. Aim: This study was done to evaluate recombinant tissue plasminogen activator r-tpa in acute ischemic stroke in hyper acute phase, in selected patients of western Utter Pradesh, in terms of feasibility and effectivity. Design: Open, non randomized study. Materials and Methods: Thirty two patients were classified using Trial of ORG 10172 in Acute Stroke treatment (TOAST criteria (large artery atherosclerotic = 8; cardio embolic = 6; small vessel occlusion = 14; other determined etiology = 2; undetermined etiology = 2. The mean time to reach the hospital was 2 h (1.15-3.0, the mean door to CT scan 20 min (10-40 and door to r-tpa injection was 30 min (24-68. The National Institute of Health Stroke Scale (NIHSS scores ranged from 11-22 (mean 15.5 +2.7. The dose of r-tpa administered was 0.9 mg/kg. Results: Twenty one patients (65.6% showed significant improvement on the NIHSS score, at 48 h (4 points. (Mean change = 10; range = 4-17. At one month, 25 (78% recorded improvement on the Barthel index (mean change = 45%. One developed frontal lobe hemorrhage and another developed recurrent stroke; one died of aspiration; and four showed no improvement. Modified Rankin score (m RS was administered at the end of three months to 28 patients (90%; however, the rest could not be directly observed. The average modified Rankin Score was 1.2 (0-2. Conclusions: Hyperacute thrombolysis was found feasible and effective in selected patients with AIS from western Utter Pradesh and who had poor affordability.

  2. Heterologous recombinant protein with decapacitating activity prevents and reverts cryodamage in ram sperm: An emerging biotechnological tool for cryobiology.

    Zalazar, L; Ledesma, A; Hozbor, F; Cesari, A

    2016-01-01

    During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize. PMID

  3. 1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

    The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka ∼ 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degree C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5

  4. Recombinant activated factor VII in the treatment of intractable non-surgical bleeding following major vascular procedures

    Končar Igor B.

    2008-01-01

    Full Text Available INTRODUCTION A recombinant form of activated factor VII (rFVIIa is a haemostatic drug that is approved for use in haemophiliacs with antibodies to factor VIII or factor IX. Most recent studies and clinical experience have shown that rFVIIa (NovoSeven ®, Novo Nordisk A/S, Denmark gives extreme haemostatic effect in patients with severe "non-haemophilic" bleeding produced after trauma and major surgery. OBJECTIVE We present our preliminary experience of the use of rFVIIa in vascular surgery when conventional haemostatic measures are inadequate. METHOD There were 32 patients divided into five groups: Group I - 14 patients with ruptured abdominal aortic aneurysms; Group II - 10 patients with thoracoabdominal aortic aneurysms; Group III - 5 patients with retroperitoneal tumors involving great abdominal vessels; Group IV - 2 patients with portal hypertension and Group V - one patient with iatrogenic injury of brachial artery and vein during fibrinolytic treatment, because of myocardial infarction. RESULTS Clinical improvement was detected following treatment in 29 patients. Bleeding was successfully controlled as evidenced by improved haemodynamic parameters and decreased inotropic and transfusion requirements. CONCLUSION In vascular patients more liberal use of rFVIIa is limited, because no randomized controlled trial has proved its efficacy and safety in such patients; while also keeping in mind that the price of a 4.8 mg of rFVIIa is $4,080. We recommend the use of rFVIIa in vascular surgery only during and after operative treatment of thoracoabdominal aortic aneurysms, ruptured abdominal aortic aneurysms, retroperitoneal tumors involving the aorta and/or inferior vena cava, as well as portal hypertension, when non-surgical massive uncontrolled bleeding are present.

  5. Subretinal recombinant tissue plasminogen activator and pneumatic displacement for the management of subretinal hemorrhage occurring after anti-VEGF injections for wet AMD

    Tognetto, D

    2011-01-01

    Daniele Tognetto, Eirini Skiadaresi, Paolo Cecchini, Giuseppe RavalicoEye Clinic, University of Trieste, Trieste, ItalyAbstract: We describe three cases of submacular hemorrhage that occurred two to four days after anti-VEGF intravitreal injection for occult choroidal neovascularisation in age-related macular degeneration and their management with 25 gauge pars plana vitrectomy with injection of subretinal recombinant tissue plasminogen activator (rTPA) followed by fluid-air exchange and post...

  6. Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line

    Kuwana, Yoshihisa; Funayama, Kikuko; Miyaji, Hiromasa; Hasegawa, Mamoru; Yoshida, Hajime; Itoh, Seiga

    2001-01-01

    The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of ∼40 mg ml-1 in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the disruption of expression...

  7. The therapeutic effect and prognosis of acute cerebral infarction patients with atrial fibrillation treated by intravenous thrombolysis with recombinant tissue plasminogen activator

    尤寿江

    2013-01-01

    Objective To investigate the efficacy and safety of intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA) in acute cerebral infarct patients with atrial fibrillation (AF) and the predicting factors of poor prognosis.Methods Totally 162 patients with acute cerebral infarct were treated with rt-PA within 4.5hours from the onset.According to past history and the electrocardiogram,the patients was classified into AF

  8. Cytoplasmic expression of recombinant interleukin-2 and interleukin-4 proteins results in hydrogen peroxide accumulation and reduction in catalase activity in Escherichia coli

    M.S Hejazi; F Karimi; E Mehdizadeh Aghdam; Barzegari, A. (MSc); M Farshdosti Hagh; Parvizi, M.; L Mahmoodi Azar; Hejazi, M. A.

    2009-01-01

    Background and the purpose of the study: The Reactive oxygen species (ROS) is induced in the cells following various stresses but the effect of recombinant protein expression on ROS generation has not been studied yet. In this study, H2O2 concentration and catalase activity variations and their correlation with cell growth following cytoplasmic expression of human interleukin-2 (hIL-2) and mouse interleukin-4 (mIL-4) in Escherichia coli were investigated. Additionally, the effect of recombina...

  9. Influence of Foreign DNA Introduction and Periplasmic Expression of Recombinant Human Interleukin-2 on Hydrogen Peroxide Quantity and Catalase Activity in Escherichia coli

    Lena Mahmoudi Azar; Elnaz Mehdizadeh Aghdam; Farrokh Karimi; Babak Haghshenas; Abolfazl Barzegari; Parichehr Yaghmaei; Mohammad Saeid Hejazi

    2013-01-01

    Purpose: Oxidative stress is generated through imbalance between composing and decomposing of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Effect of cytoplasmic recombinant protein expression on Hydrogen peroxide concentration and catalase activity was previously reported. In comparison with cytoplasm, periplasmic space has different oxidative environment. Therefore, in present study we describe t...

  10. The Recombinant Bacteriophage Endolysin HY-133 Exhibits In Vitro Activity against Different African Clonal Lineages of the Staphylococcus aureus Complex, Including Staphylococcus schweitzeri.

    Idelevich, Evgeny A; Schaumburg, Frieder; Knaack, Dennis; Scherzinger, Anna S; Mutter, Wolfgang; Peters, Georg; Peschel, Andreas; Becker, Karsten

    2016-04-01

    HY-133 is a recombinant bacteriophage endolysin with bactericidal activity againstStaphylococcus aureus Here, HY-133 showedin vitroactivity against major African methicillin-susceptible and methicillin-resistantS. aureuslineages and ceftaroline/ceftobiprole- and borderline oxacillin-resistant isolates. HY-133 was also active againstStaphylococcus schweitzeri, a recently described species of theS. aureuscomplex. The activity of HY-133 on the tested isolates (MIC50, 0.25 μg/ml; MIC90, 0.5 μg/ml; range, 0.125 to 0.5 μg/ml) was independent of the species and strain background or antibiotic resistance. PMID:26833148

  11. A soil-column gas chromatography (SCGC) approach to explore the thermal desorption behavior of hydrocarbons from soils.

    Yu, Ying; Liu, Liang; Shao, Ziying; Ju, Tianyu; Sun, Bing; Benadda, Belkacem

    2016-01-01

    A soil-column gas chromatography approach was developed to simulate the mass transfer process of hydrocarbons between gas and soil during thermally enhanced soil vapor extraction (T-SVE). Four kinds of hydrocarbons-methylbenzene, n-hexane, n-decane, and n-tetradecane-were flowed by nitrogen gas. The retention factor k' and the tailing factor T f were calculated to reflect the desorption velocities of fast and slow desorption fractions, respectively. The results clearly indicated two different mechanisms on the thermal desorption behaviors of fast and slow desorption fractions. The desorption velocity of fast desorption fraction was an exponential function of the reciprocal of soil absolute temperature and inversely correlated with hydrocarbon's boiling point, whereas the desorption velocity of slow desorption fraction was an inverse proportional function of soil absolute temperature, and inversely proportional to the log K OW value of the hydrocarbons. The higher activation energy of adsorption was found on loamy soil with higher organic content. The increase of carrier gas flow rate led to a reduction in the apparent activation energy of adsorption of slow desorption fraction, and thus desorption efficiency was significantly enhanced. The obtained results are of practical interest for the design of high-efficiency T-SVE system and may be used to predict the remediation time. PMID:26335523

  12. Adsorption, reaction and desorption of hydrogen on modified Pd(1 1 1) surfaces

    The interaction of hydrogen (deuterium) with different modified Pd(1 1 1) surfaces has been investigated. The focus was put on the energy and angel dependence of the desorbing molecules from oxygen covered, potassium covered and vanadium oxide covered surfaces. Conventional adsorption/desorption as well as permeation/desorption experiments were performed. For the oxygen covered surface optimum reaction rates for water production and the energy distribution of the reaction products were determined, both for the reaction of oxygen with molecular hydrogen as well as with atomic hydrogen. Potassium on the surface enhances the activation barrier for hydrogen adsorption resulting in a hyper-thermal desorption flux and a forward focused angular distribution of desorption. Permeation/desorption of deuterium from ultra-thin vanadium oxide films yield mainly thermalized desorbing molecules or slightly hyper-thermal, depending on the oxidation state of the surface oxide.

  13. Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the peroxisome proliferator-activated receptor-γ

    LIU Ming; WANG Yi-sheng; LI Yue-bai; ZHAO Guo-qiang

    2012-01-01

    Background Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease,with a high disability rate.At present,efficient prevention and treatment of steroid-induced ONFH is still lacking.The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH.RNA interference (RNAi) is a tool for functional gene analysis,which has been successfully used to down-regulate the levels of specific target proteins.Therefore,down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.Methods According to the principles of siRNA design,three duplex siRNA sequences (971-989,1253-1271 and 1367-1385) derived from the PPARy gene (NM_001082148) were synthesized.These duplexes were annealed,purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector.The shuttle vector was transfected into HEK293 cells.The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.Results After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences,products were identified by gel electrophoresis.These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367 were constructed.These sequences of these recombinant vectors were confirmed.We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ.After purification,the virus titer was higher than 1010 plaque forming unit (PFU)/ml.Conclusion In this study,three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ,including shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367,were successfully constructed and high titers of recombinant adenovirus were obtained.

  14. Influence of soil properties on heavy metal sequestration by biochar amendment: 2. Copper desorption isotherms.

    Uchimiya, Minori; Klasson, K Thomas; Wartelle, Lynda H; Lima, Isabel M

    2011-03-01

    Contaminant desorption constrains the long-term effectiveness of remediation technologies, and is strongly influenced by dynamic non-equilibrium states of environmental and biological media. Information is currently lacking in the influence of biochar and activated carbon amendments on desorption of heavy metal contaminants from soil components. In this study, copper sorption-desorption isotherms were obtained for clay-rich, alkaline San Joaquin soil with significant heavy metal sorption capacity, and eroded, acidic Norfolk sandy loam soil having low capacity to retain copper. Acidic pecan shell-derived activated carbon and basic broiler litter biochar were employed in desorption experiments designed to address both leaching by rainfall and toxicity characteristics. For desorption in synthetic rain water, broiler litter biochar amendment diminished sorption-desorption hysteresis. In acetate buffer (pH 4.9), significant copper leaching was observed, unless acidic activated carbon (pH(pzc)=3.07) was present. Trends observed in soluble phosphorus and zinc concentrations for sorption and desorption equilibria suggested acid dissolution of particulate phases that can result in a concurrent release of copper and other sorbed elements. In contrast, sulfur and potassium became depleted as a result of supernatant replacements only when amended carbon (broiler litter biochar) or soil (San Joaquin) contained appreciable amounts. A positive correlation was observed between the equilibrium aluminum concentration and initial copper concentration in soils amended with acidic activated carbon but not basic biochar, suggesting the importance of cation exchange mechanism, while dissolution of aluminum oxides cannot be ruled out. PMID:21190718

  15. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  16. Influence of Foreign DNA Introduction and Periplasmic Expression of Recombinant Human Interleukin-2 on Hydrogen Peroxide Quantity and Catalase Activity in Escherichia coli

    Lena Mahmoudi Azar

    2013-08-01

    Full Text Available Purpose: Oxidative stress is generated through imbalance between composing and decomposing of reactive oxygen species (ROS. This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Effect of cytoplasmic recombinant protein expression on Hydrogen peroxide concentration and catalase activity was previously reported. In comparison with cytoplasm, periplasmic space has different oxidative environment. Therefore, in present study we describe the effect of periplasmic expression of recombinant human interleukin-2 (hIL-2 on H2O2 concentration and catalase activity in Escherichia coli and their correlation with cell growth. Methods: Having constructed pET2hIL2 vector, periplasmic expression of hIL-2 was confirmed. Then, H2O2 concentration and catalase activity were determined at various ODs. Wild type and empty vector transformed cells were used as negative controls. Results: It was shown that H2O2 concentration in hIL-2 expressing cells was significantly higher than its concentration in wild type and empty vector transformed cells. Catalase activity and growth rate reduced significantly in hIL-2 expressing cells compared to empty vector transformed and wild type cells. Variation of H2O2 concentration and catalase activity is intensive in periplasmic hIL-2 expressing cells than empty vector containing cells. Correlation between H2O2 concentration elevation and catalase activity reduction with cell growth depletion are also demonstrated. Conclusion: Periplasmic expression of recombinant hIL-2 elevates the host cell’s hydrogen peroxide concentration possibly due to reduced catalase activity which has consequent suppressive effect on growth rate.

  17. Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

    Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

    1992-06-01

    In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

  18. Generalized multibody computer simulations of plasma-wall desorption and energy-transfer processes

    We present calculations that describe the kinematic factors influencing the recombinant desorption of hydrogen from metal surfaces and a preliminary study of the sputtering and possible recombination of subsurface hydrogen following surface impact of a flux of ions. A new multi-body computer molecular dynamics method used in these studies is discussed. In the first instance we are able to describe both a scattering channel in which backscattering is the dominant result and also a reactive channel leading to molecular formation with the molecules leaving importance of these processes to negative ion formation is discussed

  19. Superior hydrogen absorption and desorption behavior of Mg thin films

    Qu, Jianglan; Wang, Yuntao; Xie, Lei; Zheng, Jie; Liu, Yang; Li, Xingguo

    Pd-capped Mg films prepared by magnetron sputtering achieved complete dehydrogenation in air at room temperature and behaved as favorable gasochromic switchable mirrors. Their cyclic hydrogen absorption and desorption kinetics in air were investigated by using the Bruggeman effective medium approximation. The overall activation energy was 80 kJ mol -1, while the reaction orders controlling desorption were deduced to be n = 2 at 328 K and n = 1 at lower temperatures by analyzing the transmittance data. The hydrogen diffusion coefficient and the corresponding activation energy were calculated by electrochemical measurements. Mg thin films exhibited the smaller activation energy and remarkable diffusion kinetics at room temperature which implied potential applications in smart windows.

  20. Use of activated recombinant factor VII for severe coagulopathy post ventricular assist device or orthotopic heart transplant

    Despotis George J

    2007-07-01

    Full Text Available Abstract Background Ventricular assist devices(VAD implantation/removal is a complex surgical procedure with perioperative bleeding complications occurring in nearly half of the cases. Recombinant activated factor VII (rFVIIa has been used off-label to control severe hemorrhage in surgery and trauma. We report here our experience with rFVIIa as a rescue therapy to achieve hemostasis in patients undergoing orthotopic heart transplant (OHT and/or VAD implantation. Methods A retrospective review was conducted from Jan 03 to Aug 05 for patients who received rFVIIa for the management of intractable bleeding unresponsive to standard hemostatic blood component therapy. Blood loss and the quantity of blood products, prior to, and for at least 12 hours after, administration of rFVIIa were recorded. Results Mean patient age was 53, (38–64 yrs, mean dose of rFVIIa administered was 78.3 μg/kg (24–189 μg/kg in 1–3 doses. All patients received the drug either intraoperatively or within 6 hours of arrival in ICU. Mean transfusion requirements and blood loss were significantly reduced after rFVIIa administration (PRBC's; 16.9 ± 13.3 to 7.1 ± 6.9 units, FFP; 13.1 ± 8.2 to 4.1 ± 4.9 units, platelets; 4.0 ± 2.8 to 2.1 ± 2.2 units, p Conclusion In this review, there was a significant decrease in transfusion requirement and blood loss after rFVIIa administration. Although, 5/17 developed thromboembolic complications, these patients may have been at higher risk based on the multiple modality therapy used to manage intractable bleeding. Nevertheless, the exact role of rFVIIa with respect to development of thromboembolic complications cannot be clearly determined. Further investigation is needed to determine rFVIIa's safety and its effectiveness in improving postoperative morbidity and mortality.

  1. Intravenous thrombolysis with recombinant tissue plasminogen activator for ischemic stroke patients over 80 years old: the Fukuoka Stroke Registry.

    Ryu Matsuo

    Full Text Available The benefit of intravenous recombinant tissue plasminogen activator (rt-PA therapy for very old patients with acute ischemic stroke remains unclear. The aim of this study was to elucidate the efficacy and safety of intravenous rt-PA therapy for patients over 80 years old.Of 13,521 stroke patients registered in the Fukuoka Stroke Registry in Japan from June 1999 to February 2013, 953 ischemic stroke patients who were over 80 years old, hospitalized within 3 h of onset, and not treated with endovascular therapy were included in this study. Among them, 153 patients were treated with intravenous rt-PA (0.6 mg/kg. For propensity score (PS-matched case-control analysis, 148 patients treated with rt-PA and 148 PS-matched patients without rt-PA therapy were selected by 1:1 matching with propensity for using rt-PA. Clinical outcomes were neurological improvement, good functional outcome at discharge, in-hospital mortality, and hemorrhagic complications (any intracranial hemorrhage [ICH], symptomatic ICH, and gastrointestinal bleeding.In the full cohort of 953 patients, rt-PA use was associated positively with neurological improvement and good functional outcome, and negatively with in-hospital mortality after adjustment for multiple confounding factors. In PS-matched case-control analysis, patients treated with rt-PA were still at lower risk for unfavorable clinical outcomes than non-treated patients (neurological improvement, odds ratio 2.67, 95% confidence interval 1.61-4.40; good functional outcome, odds ratio 2.23, 95% confidence interval 1.16-4.29; in-hospital mortality, odds ratio 0.30, 95% confidence interval 0.13-0.65. There was no significant association between rt-PA use and risk of hemorrhagic complications in the full and PS-matched cohorts.Intravenous rt-PA therapy was associated with improved clinical outcomes without significant increase in risk of hemorrhagic complications in very old patients (aged>80 years with acute ischemic stroke.

  2. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  3. Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections.

    Kobayashi, I.; Murialdo, H; Crasemann, J M; Stahl, M. M.; Stahl, F. W.

    1982-01-01

    Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli. Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward). Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive. We surmise that chi action in phage lambda is coupled with ...

  4. Cytoplasmic expression of recombinant interleukin-2 and interleukin-4 proteins results in hydrogen peroxide accumulation and reduction in catalase activity in Escherichia coli

    M.S Hejazi

    2009-08-01

    Full Text Available Background and the purpose of the study: The Reactive oxygen species (ROS is induced in the cells following various stresses but the effect of recombinant protein expression on ROS generation has not been studied yet. In this study, H2O2 concentration and catalase activity variations and their correlation with cell growth following cytoplasmic expression of human interleukin-2 (hIL-2 and mouse interleukin-4 (mIL-4 in Escherichia coli were investigated. Additionally, the effect of recombinant protein expression under different conditions was compared to the effect of foreign DNA introduction on these factors. Methods: Plasmids pEThIL-2 and pETmIL-4 were used for expression of human interleukin-2 (hIL-2 and mouse interleukin-4 (mIL-4 inside the cytoplasm of the cells. Having confirmed protein expression using SDS-PAGE analysis and ELISA assay, H2O2 concentration and catalase activity were measured at various ODs. Results and major conclusion: Empty vector introduction increased significantly H2O2 concentration of the cells. However, H2O2 concentration in hIL-2 and mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells. Catalase activity was reduced in foreign DNA introduced cells. It was more lowered following expression of recombinant proteins. Results of this study revealed the relationship between foreign DNA introduction and protein expression with H2O2 elevation and catalase activity reduction. There was also correlation between H2O2 elevation and reduction in catalase activity with the cell growth depression.

  5. Desorption and sublimation kinetics for fluorinated aluminum nitride surfaces

    King, Sean W., E-mail: sean.king@intel.com; Davis, Robert F. [Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695 (United States); Nemanich, Robert J. [Department of Physics, North Carolina State University, Raleigh, North Carolina 27695 (United States)

    2014-09-01

    The adsorption and desorption of halogen and other gaseous species from surfaces is a key fundamental process for both wet chemical and dry plasma etch and clean processes utilized in nanoelectronic fabrication processes. Therefore, to increase the fundamental understanding of these processes with regard to aluminum nitride (AlN) surfaces, temperature programmed desorption (TPD) and x-ray photoelectron spectroscopy (XPS) have been utilized to investigate the desorption kinetics of water (H{sub 2}O), fluorine (F{sub 2}), hydrogen (H{sub 2}), hydrogen fluoride (HF), and other related species from aluminum nitride thin film surfaces treated with an aqueous solution of buffered hydrogen fluoride (BHF) diluted in methanol (CH{sub 3}OH). Pre-TPD XPS measurements of the CH{sub 3}OH:BHF treated AlN surfaces showed the presence of a variety of Al-F, N-F, Al-O, Al-OH, C-H, and C-O surfaces species in addition to Al-N bonding from the AlN thin film. The primary species observed desorbing from these same surfaces during TPD measurements included H{sub 2}, H{sub 2}O, HF, F{sub 2}, and CH{sub 3}OH with some evidence for nitrogen (N{sub 2}) and ammonia (NH{sub 3}) desorption as well. For H{sub 2}O, two desorption peaks with second order kinetics were observed at 195 and 460 °C with activation energies (E{sub d}) of 51 ± 3 and 87 ± 5 kJ/mol, respectively. Desorption of HF similarly exhibited second order kinetics with a peak temperature of 475 °C and E{sub d} of 110 ± 5 kJ/mol. The TPD spectra for F{sub 2} exhibited two peaks at 485 and 585 °C with second order kinetics and E{sub d} of 62 ± 3 and 270 ± 10 kJ/mol, respectively. These values are in excellent agreement with previous E{sub d} measurements for desorption of H{sub 2}O from SiO{sub 2} and AlF{sub x} from AlN surfaces, respectively. The F{sub 2} desorption is therefore attributed to fragmentation of AlF{sub x} species in the mass spectrometer ionizer. H{sub 2} desorption exhibited

  6. Kinetics of volatile fluoride desorption from granular sodium fluoride. Part II. Desorption of WF6, MoF6, and HF

    Desorption of tungsten hexafluoride, molybdenum hexafluoride and hydrogen fluoride sorbed on granulated sodium fluoride was studied in the temperature ranges 563-583 K, 463-483 K and 460-490 K respectively. It was ascertained that the process of WF6 and MoF6 desorption is limited by diffusion of the sorbate molecules from the bulk of sodium fluoride granules and it takes place in two stages. Chemical reaction of NaF · HF complex decomposition is the limiting stage of HF desorption. The values of apparent activation energy of WF6, MoF6 and HF desorption amounted to 95200 ± 2000 J/mol, 55000 ± 2500 J/mol and 88500 ± 1500 J/mol, respectively

  7. High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

    Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

    2016-08-01

    The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits. PMID:27230577

  8. Safety and immunogenicity of recombinant poxvirus HIV-1 vaccines in young adults on highly active antiretroviral therapy

    Greenough, Thomas C.; Cunningham, Coleen K; Muresan, Petronella; McManus, Margaret; Persaud, Deborah; Fenton, Terry; Barker, Piers; Gaur, Aditya; Panicali, Dennis; Sullivan, John L.; Luzuriaga, Katherine

    2008-01-01

    A trial to evaluate the safety and immunogenicity of recombinant modified vaccinia Ankara (MVA) and fowlpox (FP) vectors expressing multiple HIV-1 proteins was conducted in twenty HIV-1 infected youth with suppressed viral replication on HAART. The MVA and FP-based multigene HIV-1 vaccines were safe and well tolerated. Increased frequencies of HIV-1 specific CD4+ proliferative responses and cytokine secreting cells were detected following immunization. Increased frequencies and breadth of HIV...

  9. Preparation and Absorption/Desorption Performance of Gypsum-based Humidity Controlling Materials

    JIANG Hongyi; LUAN Congmei

    2011-01-01

    Absorption/desorption properties of some humidity controlling materials which contain gypsum as basic cement materials and activated Sepiolite powder as humidity controlling media were tested.The kinetics curve of moisture adsorption/desorption were drawn and humidity controlling performance in nature environment was verified.The experimental results show that moisture absorption/desorption rates are increased,and the speed is also accelerated.These materials,which can adjust environmental humidity effectively,are new ones with good humidity controlling performance.

  10. Role of Farben centers in electron- and photon-stimulated desorption from alkali halides

    Measurements of the time history of ground-state neutral lithium desorption from LiF during pulsed electron irradiation have resulted in a new model for the lithium-desorption mechanism. We show that the slow diffusion to the surface of bulk Farben (F) centers created by the electron-beam is the rate-controlling factor responsible for most of the time history of the lithium desorption rather than the thermal evaporation of the metal from the surface. Comparison between theoretical and experimental time dependencies yields values for the F center diffusion constant and its activation energy

  11. Thermal desorption of oxygen from near-stoichiometric cationic vanadium oxide clusters

    Kurokawa, Hodaka; Mafuné, Fumitaka

    2016-05-01

    Oxygen desorption from cationic vanadium oxide clusters, VnOm+ (n = 2-10), composed of a near-stoichiometric (n:m = 2:5) frame with excess oxygen attached was investigated in a thermal energy region by time-of-flight mass spectrometry and thermal desorption spectrometry. Oxygen molecules were observed to desorb from the clusters during heating. The activation energy for desorption was estimated from the temperature dependence of different clusters and exhibited an even-odd alternation with respect to the cluster size, n. This alternation can be explained in terms of oxidation states of the vanadium atoms.

  12. A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion

    Yi Ma; Tianjie Luo; Wenna Xu; Zulu Ye; An Hong

    2012-01-01

    The recombinant peptide,DBAYL,a promising therapeutic peptide for type 2 diabetes,is a new,potent,and highly selective agonist for VPAC2 generated through sitedirected mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP),vasoactive intestinal peptide (VIP),and related analogs.The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization.As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 I of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro.The bioactivity assay of DBAYL showed that it displaced [125I]PACAP38 and [125I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 ± 6.9 and 47.1 ± 4.9 nM,respectively,which were significantly lower than that of BAY55-9837,one established VPAC2 agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC5o) of 0.68 nM,whereas the receptor potency of DBAYL at human VPAC1 (ECso of 737 nM) was only 1/1083of that at human VPAC2,and DBAYL had no activity toward human PAC1 receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin receptor substrate 1 (IRS-1) and glucose transporter 4(GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes.Compared with BAY55-9837 and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice.These results suggested that DBAYL could efficiently improve glucose uptake and glucose-dependent insulin

  13. Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

    Uckun, Fatih M.; Rajamohan, Francis; Pendergrass, Sharon; Ozer, Zahide; Waurzyniak, Barbara; Mao, Chen

    2003-01-01

    A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102(151AA152) and FLP-105(191AA192) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substanti...

  14. Use of recombinant activated factor VII for reduction of perioperative blood loss during elective surgical correction of spine deformity in a Jehovah's Witness. Case report.

    Kącka, Katarzyna; Kącki, Wojciech; Merak, Joanna; Błęka, Adam

    2010-01-01

    Planned surgical procedures at patients who refuse allogenic blood transfusion because of religious convictions are important problem, not only medical but also ethical and juristical. At the study authors report the successful use of activated recombinant factor VII (rFVIIa) for the reduction of perioperative blood loss in four years old child - Jehovah's Witness, who had planned Torode kyphectomy. Applied perioperative management together with preparing to surgery with erythropoietin allowed for reduction of blood loss and avoiding of blood transfusion. Authors state, that appropriate perioperative proceeding makes a possibility of safe surgical procedures also at patients who refuse the transfusion. PMID:21057153

  15. Enhanced hydrogen desorption property of MgH2 with the addition of cerium fluorides

    Highlights: • Activation energy of MgH2 desorption is remarkably reduced with the dopant of CeF4. • The improvement might be attributed to new Ce–F–Mg species at the CeF4/MgH2 interface. • Easy electron transfer induced from the high valence Ce-cation benefits MgH2 desorption. - Abstract: Hydrogen desorption property of MgH2 doped with cerium fluorides with different valences prepared using ball milling has been studied. CeF4 is catalytically active for hydrogen desorption of MgH2. Hydrogen desorption temperature and apparent activation energy of MgH2 are significantly reduced with dopant of 2 mol% of CeF4, which might be attributed to the formation of a new Ce–F–Mg specie at the CeF4/MgH2 interface and the easy electron transfer induced from the high valence Ce-cation. The apparent activation energy of hydrogen desorption of MgH2 is reduced from ∼160 kJ/mol to ∼110 kJ/mol with the dopant of CeF4

  16. Soluble recombinant influenza vaccines.

    Fiers, W; Neirynck, S; Deroo, T; Saelens, X; Jou, W M

    2001-01-01

    Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the...

  17. Bentazone adsorption and desorption on agricultural soils

    Boivin, A.; Cherrier, R.; Schiavon, M

    2005-01-01

    Herbicide fate and transport in soils greatly depend upon adsorption-desorption processes. Batch adsorption and desorption experiments were performed with the herbicide bentazone using 13 contrasted agricultural soil samples. Bentazone was found to be weakly sorbed by the different soils, showing average Freundlich adsorption coefficients (Kf) value of 1.4 ± 2.3 mg1 - nf Lnf kg-1. Soil organic matter content did not have a significant effect on bentazone sorption (r2 = 0.12), whereas natural ...

  18. Neutrino-recoil induced desorption

    Nuclear decay induced 37Cl ion desorption from the electron capture decay 37Ar→37Cl+ν is reported for the first time. A mixture of one part 36Ar and ∼5x10-5 parts 37Ar (36/37Ar) is physisorbed on a gold-plated Si wafer kept at 16 K under ultrahigh vacuum conditions. The time of flight (TOF) of recoiled 37Cl ions is measured using coincidence techniques. The observed kinetic energy distribution of the 37Cl ions is approximately Gaussian in shape, with a maximum at ∼9.0 eV and a full width at half-maximum of ∼3 eV. Considering the binding energy of physisorbed 37Ar is ∼80 meV, the 9-eV peak energy compares well with that of the gas-phase value, where conservation of the energy and momentum fixes the kinetic energy of 37Cl ions at 9.54 eV. Using a combination of TOF and retarding field energy analysis, the charge states of detected ions for 1 ML (monolayer) of 36/37Ar are determined as 53%+1e, 21%+2e, and 26%+ne, where n≥3. The fraction of decaying 37Ar atoms which emerge from the surface as positive 37Cl ions is found to be 10%. Finally, a strong charge exchange reaction between a 37Cl ion and near-neighbor atoms causes a Coulomb explosion within the multilayers, increasing the kinetic energy of desorbing ions by as much as ∼7 eV

  19. Expression of L protein of vesicular stomatitis virus Indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro.

    Mathur, M.; Das, T.; Banerjee, A K

    1996-01-01

    The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) Indiana serotype, a multifunctional catalytic subunit of the viral RNA polymerase, has been expressed in Spodoptera frugiperda cells infected with recombinant baculovirus BacPAK6-L containing the L gene under the control of a polyhedrin promoter. The recombinant L protein was biologically active and supported viral mRNA synthesis in vitro. When the expressed L protein was purified by phosphocellulose column chromatography, it e...

  20. Relationship of Impairment of Schistosome 28-Kilodalton Glutathione S-Transferase (GST) Activity to Expression of Immunity to Schistosoma mattheei in Calves Vaccinated with Recombinant Schistosoma bovis 28-Kilodalton GST

    Grzych, Jean-Marie; De Bont, Jan; Liu, Jinli; Neyrinck, Jean-Loup; Fontaine, Josette; Vercruysse, Jozef; Capron, André

    1998-01-01

    Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) o...

  1. Deuterium desorption behavior of solid tritium breeding material, lithium titanate

    Several types of blanket module, solid breeder/water or helium cooling, LiPb breeder/helium cooling, liquid LiN and Flibe, have been developed toward both ITER and a demonstration reactor in Japan. In the solid breeder blanket cooled by water, pellets of Li2TiO3 will be employed as tritium breeding material. Structure material in this blanket is low activation ferritic steel, F82H. The operation temperature is limited below approximately 820 K owing to swelling caused by neutron irradiation. Tritium produced by fusion neutrons in this breeding material has to be desorbed under a blanket operation for tritium recovery to be easy. The blanket module, however, has a spatial distribution of temperature. Thus, the tritium desorption behavior has to be clarified in order to make a scheme for tritium recovery. In the present study, a solid breeding material, Li2TiO3, was irradiated by 1.7 keV deuterium ions, and an amount of retained deuterium and deuterium desorption behavior were investigated using a thermal desorption. Dependence of deuterium fluence on amount of retained deuterium was also obtained. In order to examine trapping mechanisms of deuterium in Li2TiO3, similar experiments were conducted for Li and Ti. Deuterium implanted to Li2TiO3 desorbed in forms of HD, D2, HDO and D2O. The amount of deuterium desorbed in form of HD was a few times larger than those of other gas species. The desorption peak appeared at 600 K, but significant desorption up to 900 K was observed. The range of temperature in the lithium titanate of the blanket module is assumed from 550 K to 1200 K. These results suggest that the tritium produced in the blanket is partly not desorbed. Thus, the spatial distribution of temperature in the blanket has to be controlled for the tritium to be desorbed during the operation. The desorption spectra of deuterium in Li2TiO3 were similar to those of Li. This suggests that most of implanted deuterium is trapped in forms of Li-D and Li-OD. Based upon the

  2. Cloning, Expression and Activity of Recombinant Human Angiostatin%重组人血管抑素的克隆、表达及活性测定

    姜颖; 孙陆果; 于永利

    2000-01-01

    目的:研究血管抑素(angiostatin)的临床应用价值,将其开发为多肽类药物。方法:以RT-PCR从胎儿肝脏钓取人血管抑素全编码区cDNA,使用Pichia分泌型酵母表达系统表达重组人血管抑素,重组蛋白经肝素亲和层析纯化后,以鸡胚尿囊膜法血管生成实验及小鼠创伤愈合实验检测活性。结果:重组人血管抑素在酵母系统中得到较高量表达(5 mg/L),经肝素亲和层析纯化,SDS-PAGE显示分子量为43 kD。活性实验表明,重组蛋白能够抑制新生血管形成、抑制伤口愈合。结论:重组人血管抑素在酵母系统中实现高效表达,并具有很好的生物学活性。%Objective: To express recombinant human angiostatin for further application in clinic. Methods: The complete encoding eDNA of human angiostatin was isolated from human embryo liver with RT-PCR and expressed in secretory Pichia expression system. Recombinant human angiostatin was purified with heparin sepharose chromatography and its activity was determined in chick embryo chorioallantoic membrane (CAM) and wound healing assays. Results: Expressed in large quantity (yield=5 mg/L) and purified with heparin sepharose, recombinant angiostatin was showed to have a molecular weight of 43 kD in SDS-PAGE and potently inhibit angiogenesis and wound healing. Conclusion: Recombinant human angiostatin was expressed efficiently in a biologically active form.

  3. A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

    Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

    2014-03-01

    Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

  4. Modelling of discrete TDS-spectrum of hydrogen desorption

    Rodchenkova, Natalia I.; Zaika, Yury V.

    2015-12-01

    High concentration of hydrogen in metal leads to hydrogen embrittlement. One of the methods to evaluate the hydrogen content is the method of thermal desorption spectroscopy (TDS). As the sample is heated under vacuumization, atomic hydrogen diffuses inside the bulk and is desorbed from the surface in the molecular form. The extraction curve (measured by a mass-spectrometric analyzer) is recorded. In experiments with monotonous external heating it is observed that background hydrogen fluxes from the extractor walls and fluxes from the sample cannot be reliably distinguished. Thus, the extraction curve is doubtful. Therefore, in this case experimenters use discrete TDS-spectrum: the sample is removed from the analytical part of the device for the specified time interval, and external temperature is then increased stepwise. The paper is devoted to the mathematical modelling and simulation of experimental studies. In the corresponding boundary-value problem with nonlinear dynamic boundary conditions physical- chemical processes in the bulk and on the surface are taken into account: heating of the sample, diffusion in the bulk, hydrogen capture by defects, penetration from the bulk to the surface and desorption. The model aimed to analyze the dynamics of hydrogen concentrations without preliminary artificial sample saturation. Numerical modelling allows to choose the point on the extraction curve that corresponds to the initial quantity of the surface hydrogen, to estimate the values of the activation energies of diffusion, desorption, parameters of reversible capture and hydride phase decomposition.

  5. New perspectives in vacuum high voltage insulation. II. Gas desorption

    Diamond, W T

    1998-01-01

    An examination has been made of gas desorption from unbaked electrodes of copper, niobium, aluminum, and titanium subjected to high voltage in vacuum. It has been shown that the gas is composed of water vapor, carbon monoxide, and carbon dioxide, the usual components of vacuum outgassing, plus an increased yield of hydrogen and light hydrocarbons. The gas desorption was driven by anode conditioning as the voltage was increased between the electrodes. The gas is often desorbed as microdischarges-pulses of a few to hundreds of microseconds-and less frequently in a more continuous manner without the obvious pulsed structure characteristic of microdischarge activity. The quantity of gas released was equivalent to many monolayers and consisted mostly of neutral molecules with an ionic component of a few percent. A very significant observation was that the gas desorption was more dependent on the total voltage between the electrodes than on the electric field. It was not triggered by field-emitted electrons but oft...

  6. Temperature Effect on Boron Adsorption—Desorption Kinetics in Soils

    ZHUDUANWEI; SHILEI; 等

    1999-01-01

    The effect of temperature on the properties of boron adsorption-desorption in brown-red soil,yellowbrown soil and calcareous alluvial soil of Hubei Province was investigated with the mobile displacement technique.The experimental data of B adsorption-desorption amounts and reaction time at 25 and 40℃ were fitted by the zero-order,first-order and parabolic diffusion kinetic equations.The adsorption process was in conformity with the parabolic diffusion law at both the temperatures,and the values of rate constant of the parabolic diffusion equation in B adsorption were 0.138,0.124 and 0.105 mg kg-1 min-1/2 at 25℃,and 0.147,0.146and 0.135mg kg-1 min1/2 at 40℃ for the brown-red soil,yellow-brown soil,and calcareous alluvial soil,respectively,The relationship between amount of B desorption and reaction time could be well described by the first-order kinetic equation,and the corresponding values of rate constant were 0.0422,0.0563 and 0.0384min-1 at 25℃,and 0.0408,0.0423 and 0.0401min-1 at 40℃ for the brown-red soil,the yellow-brown soil and the calcareous alluvial soil,respectively.Therefore,the desorption process of B might be related to the amount of B adsorbed in soil,The higher th temperature,the lower the amount of B adsorption of the same soil in the same reaction time,The values of the apparent activation energy of B adsorption in the three soils calculated with the rate constants of parabolic diffusion equation were 3.27,8.44 and 12.99 kJ mol-1,respectively,based on the experimental data of B adsorption amounts and reaction time at and 40℃.

  7. Gas desorption during friction of amorphous carbon films

    Rusanov, A.; Fontaine, J.; Martin, J.-M.; Mogne, T. L.; Nevshupa, R.

    2008-03-01

    Gas desorption induced by friction of solids, i.e. tribodesorption, is one of the numerous physical and chemical phenomena, which arise during friction as result of thermal and structural activation of material in a friction zone. Tribodesorption of carbon oxides, hydrocarbons, and water vapours may lead to significant deterioration of ultra high vacuum conditions in modern technological equipment in electronic, optoelectronic industries. Therefore, knowledge of tribodesorption is crucial for the performance and lifetime of vacuum tribosystems. Diamond-like carbon (DLC) coatings are interesting materials for vacuum tribological systems due to their high wear resistance and low friction. Highly hydrogenated amorphous carbon (a-C:H) films are known to exhibit extremely low friction coefficient under high vacuum or inert environment, known as 'superlubricity' or 'superlow friction'. However, the superlow friction period is not always stable and then tends to spontaneous transition to high friction. It is supposed that hydrogen supply from the bulk to the surface is crucial for establishing and maintaining superlow friction. Thus, tribodesorption can serve also as a new technique to determine the role of gases in superlow friction mechanisms. Desorption of various a-C:H films, deposited by PECVD, ion-beam deposition and deposition using diode system, has been studied by means of ultra-high vacuum tribometer equipped with a mass spectrometer. It was found that in superlow friction period desorption rate was below the detection limit in the 0-85 mass range. However, transition from superlow friction to high friction was accompanied by desorption of various gases, mainly of H2 and CH4. During friction transition, surfaces were heavily damaged. In experiments with DLC films with low hydrogen content tribodesorption was significant during the whole experiment, while low friction was not observed. From estimation of maximum surface temperature during sliding contact it was

  8. Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution.

    Gelato, Kathy A; Martin, Shelley S; Baldwin, Enoch P

    2005-11-25

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to

  9. Krypton-85 enrichment by adsorption-desorption process

    The use of activated charcoal columns in conjunction with cryogenic distillation system for concentration of krypton-85 in fuel reprocessing process off-gas stream is reported. Dynamic adsorption of krypton on activated charcoals and its subsequent desorption by applying vacuum were studied. The possible reduction in the quantity of carrier gas to be liquified in the cryogenic system by utilising this process has been discussed on the basis of results of laboratory evaluations. The possibility of elimination of air and oxygen to avoid explosion hazards associated with radiolytic formation and concentration of ozone has also been considered. (author)

  10. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines. PMID:24303205

  11. Recombinant Collagenlike Proteins

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  12. Recombinant Technology and Probiotics

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  13. Adsorption and Desorption of Methiopyrsulfuron in Soils

    WU Chun-Xian; WANG Jin-Jun; ZHANG Su-Zhi; ZHANG Zhong-Ming

    2011-01-01

    Methiopyrsulfuron is a new low-rate sulfonylurea herbicide for weed control in wheat; however, there is a lack of published information on its behavior in soils. In this study, methiopyrsulfuron adsorption and desorption were measured in seven soils sampled from Heilongjiang, Shandong, Jiangxi, Sichuan, Anhui, and Chongqing provinces of China using a batch equilibrium method. The Freundlich equation was used to described its adsorption and desorption. Adsorption isotherms were nonlinear with the values of Kf-ads, the Freundlich empirical constant indicative of the adsorption capacity,ranging from 0.75 to 2.46, suggesting that little of this herbicide was adsorbed by any of the seven soils. Soil pH and organic matter content (OM) were the main factors influencing adsorption; adsorption was negatively correlated with pH and positively correlated with OM. Methiopyrsulfuron desorption was hysteretic on the soils with high OM content and low pH.

  14. Desorption of 137Cs+ from mosses

    OLGICA NEDIC

    2002-09-01

    Full Text Available Mosses are biomonitors that accumulate large amounts of various pollutants, including radionuclides. In this work we investigated the possibility of 137Cs extraction from mosses, as well as the significance of species specificity on the efficiency of 137Cs desorption. Salt and acid solutions were used as extraction media. It was shown that a 5 % solution of both ammonium oxalate and phosphoric acid was able to desorb 81.8 % of 137Cs+ from Homalothecium sericeum, which was 39.9 % more than desorption from water. At the same time, most of the desorbed 137Cs+ was incorporated in crystals that precipitated from the solution. An interspecies difference in respect to 137Cs+ desorption was noticed.

  15. Desorption studies of adsorbed H2,on W (poly) with some surface analysis techniques

    Experimental results of H2 desorption from W (poly), with Programed Thermal Desorption, Electron Stimulated Desorption and Variations of Work Function techniques, is presented. Data analysis reveals four differents desorption states. Desorption energies, calculated from data of the three techniques, shows good agreement between them. Some desorption states of H2 present no ion production, with ESD, during desorption. (author)

  16. Recombination efficiency of molecular hydrogen on interstellar grains - II: A numerical study

    Knowledge of the recombination time on the grain surfaces has been a major obstacle in deciding the production rate of molecular hydrogen and other molecules in the interstellar medium. We present a numerical study to compute this time for molecular hydrogen for various cloud and grain parameters. We also find the time dependence, particularly when a grain is freshly injected into the system. Apart from the fact that the recombination times seem to be functions of the grain parameters such as the activation barrier energy, temperature etc., our result also shows the dependence on the number of sites in the grain S and the effective accretion rate per site as of atomic hydrogen. To put simply the average time that a pair of atomic hydrogens will take to produce one molecular hydrogen depends on how heavily the grain is already populated by atomic and molecular hydrogens and how fast the hopping and desorption times are. We show that if we write the average recombination time as Tr ∼ Sα/AH, where, AH is the hopping rate, then α could be much greater than 1 for all astrophysically relevant accretion rates. Thus the average formation rate of H2 is also dependent on the grain parameters, temperature and the accretion rate. We believe that our results will affect the overall rate of the formation of complex molecules such as methanol which requires successive hydrogenation on the grain surfaces in the interstellar medium. (author)

  17. Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

    Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

  18. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

  19. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Rui-Li Zhang

    Full Text Available The recombinant Treponema pallidum protein Tp0965 (rTp0965, one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

  20. Photoinduced Br Desorption from CsBr Thin Films Grown on Cu(100)

    Halliday, Matthew T.; Joly, Alan G.; Hess, Wayne P.; Shluger, AL

    2015-10-22

    Thin films of CsBr deposited onto metals such as copper are potential photocathode materials for light sources and other applications. We investigate desorption dynamics of Br atoms from CsBr films grown on insulator (KBr, LiF) and metal (Cu) substrates induced by sub-bandgap 6.4 eV laser pulses. The experimental results demonstrate that the peak kinetic energy of Br atoms desorbed from CsBr/Cu films is much lower than that for the hyperthermal desorption from CsBr/LiF films. Kelvin probe measurements indicate negative charge at the surface following Br desorption from CsBr/Cu films. Our ab initio calculations of excitons at CsBr surfaces demonstrate that this behavior can be explained by an exciton model of desorption including electron trapping at the CsBr surface. Trapped negative charges reduce the energy of surface excitons available for Br desorption. We examine the electron-trapping characteristics of low-coordinated sites at the surface, in particular, divacancies and kink sites. We also provide a model of cation desorption caused by Franck-Hertz excitation of F centers at the surface in the course of irradiation of CsBr/Cu films. These results provide new insights into the mechanisms of photoinduced structural evolution of alkali halide films on metal substrates and activation of metal photocathodes coated with CsBr.

  1. BEAM PIPE DESORPTION RATE IN RHIC.

    HUANG, H.; FISCHER, W.; HE, P.; HSEUH, H.C.; IRISO, U.; PTITSYN, V.; TRBOJEVIC, D.; WEI, J.; YANG, S.Y.

    2006-06-23

    In the past, an increase of beam intensity in RHIC has caused several decades of pressure rises in the warm sections during operation. This has been a major factor limiting the RHIC luminosity. About 430 meters of NEG coated beam pipes have been installed in the warm sections to ameliorate this problem. Beam ion induced desorption is one possible cause of pressure rises. A series beam studies in RHIC has been dedicated to estimate the desorption rate of various beam pipes (regular and NEG coated) at various warm sections. Correctors were used to generate local beam losses and consequently local pressure rises. The experimental results are presented and analyzed in this paper.

  2. Electronic Desorption of gas from metals

    Molvik, A W; Kollmus, H; Mahner, E; Covo, M K; Bender, M; Bieniosek, F M; Kramer, A; Kwan, J; Prost, L; Seidl, P A; Westenskow, G

    2006-11-02

    During heavy ion operation in several particle accelerators world-wide, dynamic pressure rises of orders of magnitude were triggered by lost beam ions that bombarded the vacuum chamber walls. This ion-induced molecular desorption, observed at CERN, GSI, and BNL, can seriously limit the ion beam lifetime and intensity of the accelerator. From dedicated test stand experiments we have discovered that heavy-ion induced gas desorption scales with the electronic energy loss (dE{sub e}/dx) of the ions slowing down in matter; but it varies only little with the ion impact angle, unlike electronic sputtering.

  3. STM-Induced Hydrogen Desorption via a Hole Resonance

    Stokbro, Kurt; Thirstrup, C.; Sakurai, M.;

    1998-01-01

    We report STM-induced desorption of H from Si(100)-H(2 X 1) at negative sample bias. The desorption rate exhibits a power-law dependence on current and a maximum desorption rate at -7 V. The desorption is explained by vibrational heating of H due to inelastic scattering of tunneling holes with the...... Si-H 5 sigma hole resonance. The dependence of desorption rate on current and bias is analyzed using a novel approach for calculating inelastic scattering, which includes the effect of the electric field between tip and sample. We show that the maximum desorption rate at -7 V is due to a maximum...

  4. Successful use of N-acetyl cysteine and activated recombinant factor VII in fulminant hepatic failure and massive bleeding secondary to dengue hemorrhagic fever

    Edirisooriya Maddumage Manoj

    2014-01-01

    Full Text Available Consensus on management of complicated cases of dengue infection is evolving. Dengue hemorrhagic fever (DHF occasionally progress to fulminant liver failure with high fatality rate. Inadvertent use of blood products to control massive bleeding in dengue shock syndrome may worsen fluid overload and subsequently the multi-organ dysfunction. We report a case of 37-years-old Sri Lankan man who developed fulminant liver failure and massive bleeding associated with DHF, subsequently recovered completely with supportive measures including administration of N-acetyl cysteine and activated recombinant factor VII. In conclusion, prevention of ischemic injury to liver and adoption of early aggressive supportive measures in complicated cases of dengue hemorrhagic fever is crucial for a favorable outcome. Indications for rFVIIa to arrest uncontrolled internal bleeding and use of NAC in non-acetaminophen-induced acute liver failure in complicated DHF are a platform for discussion.

  5. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  6. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations.

    Joseph A M J L Janssen

    Full Text Available To compare full-length mechano growth factor (full-length MGF with human recombinant insulin-like growth factor-I (IGF-I and human recombinant insulin (HI in their ability to activate the human IGF-I receptor (IGF-IR, the human insulin receptor (IR-A and the human insulin receptor-B (IR-B, respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR.The effects of full-length MGF, IGF-I, human mechano growth factor (MGF, Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding.IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively. However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69-1.07 and 7.83 nmol/L (95% CI: 4.87-12.58, respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold. EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69-1.84 and 73.11 nmol/L (42.87-124.69, respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64-2.57 and 35.10 nmol/L (95% 17.52-70.33, respectively.Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the physiological relevance of our in vitro findings.

  7. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; van den Dungen, Elisabeth S. R.; Thevis, Mario

    2016-01-01

    Aims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding. Results IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively. Conclusions Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the

  8. Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

    Wang, Jingxian; Xie, Ping [Donghu Experimental Station of the Lake Ecosystems, The State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Kettrup, Antonius; Schramm, Karl-Werner [GSF-National Research Centre of Environment and Health, Institute of Ecological Chemistry, Ingolstaedter Landstr. 1, D-85764 Neuherberg (Germany)

    2005-10-15

    Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit hPR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants.

  9. Distribution of trichloroacetic acid precipitated radioactivity in rabbits after intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator

    Objective: To study tissue distribution profiles of trichloroacetic acid (TCA) precipitated radioactivity in rabbits following intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator (125I-rh-sc-uPA). Methods: 125I-rh-su-uPA was prepared by Iodogen method and was purified by Saphacryl HR S-200. The radio-chemical purity was measured by reverse phase high performance liquid chromatography (RP-HPLC). Biological activity of labelled protein was measured by thrombolytic activity on fibrin plate. Results: The identification of 125I-rh-sc-uPA revealed that it was a 94.4% radio-chemically purified thrombolytic active protein. The AUC of radioactivity gradients in tissues and other specimens were as follows: kidney>urine>plasma>liver>spleen>adrenal gland>bone marrow>heart>urinary bladder>lungs>gonad>small intestinal wall>fat>lymph node>gall bladder>thymus gland>intestinal content>muscle>brain>feces in colon. Conclusion: The highest TCA precipitated radioactivity was found in plasma following I.V. of 125I-rh-u-PA, which might be favorable for the drug action. The main excretion route was urinary system

  10. Cytotoxic Effects and Osteogenic Activity of Calcium Sulfate with and without Recombinant Human Bone Morphogenetic Protein 2 and Nano-Hydroxyapatite Adjacent to MG-63 Cell Line

    Abdollah Ghorbanzadeh

    2015-10-01

    Full Text Available Objectives: The aim of this study was to assess the cytotoxic effects and osteogenic activity of recombinant human bone morphogenetic protein (rhBMP2 and nano-hydroxyapatite (n- HA adjacent to MG-63 cell line.Materials and Methods: To assess cytotoxicity, the 4,5-dimethyl thiazolyl-2,5-diphenyl tetrazolium bromide (MTT assay was used. Alkaline phosphatase (ALP activity and oste- ogenic activity were evaluated using Alizarin red and the von Kossa staining and analyzed by one-way ANOVA followed by Tukey’s post hoc test.Results: The n-HA/CS mixture significantly promoted cell growth in comparison to pure calcium sulfate (CS. Moreover, addition of rhBMP2 to CS (P=0.02 and also mixing CS with n-HA led to further increase in extracellular calcium production and ALP activity (P=0.03.Conclusion: This in vitro study indicates that a scaffold material in combination with an osteoinductive material is effective for bone matrix formation.

  11. Magnesium growth in magnesium deuteride thin films during deuterium desorption

    Checchetto, R., E-mail: riccardo.checchetto@unitn.it [Dipartimento di Fisica and CNISM, Università di Trento, Via Sommarive 14, I-38123 Trento (Italy); Miotello, A. [Dipartimento di Fisica and CNISM, Università di Trento, Via Sommarive 14, I-38123 Trento (Italy); Mengucci, P.; Barucca, G. [Dipartimento di Fisica e Ingegneria dei Materiali e del Territorio, Università Politecnica delle Marche, I-60131 Ancona (Italy)

    2013-12-15

    Highlights: ► Highly oriented Pd-capped magnesium deuteride thin films. ► The MgD{sub 2} dissociation was studied at temperatures not exceeding 100 °C. ► The structure of the film samples was analyzed by XRD and TEM. ► The transformation is controlled by the re-growth velocity of the Mg layers. ► The transformation is thermally activated, activation energy value of 1.3 ± 0.1 eV. -- Abstract: Pd- capped nanocrystalline magnesium thin films having columnar structure were deposited on Si substrate by e-gun deposition and submitted to thermal annealing in D{sub 2} atmosphere to promote the metal to deuteride phase transformation. The kinetics of the reverse deuteride to metal transformation was studied by Thermal Desorption Spectroscopy (TDS) while the structure of the as deposited and transformed samples was analyzed by X-rays diffraction and Transmission Electron Microscopy (TEM). In Pd- capped MgD{sub 2} thin films the deuteride to metal transformation begins at the interface between un-reacted Mg and transformed MgD{sub 2} layers. The D{sub 2} desorption kinetics is controlled by MgD{sub 2}/Mg interface effects, specifically the re-growth velocity of the Mg layers. The Mg re-growth has thermally activated character and shows an activation energy value of 1.3 ± 0.1 eV.

  12. Quantum theory of laser-stimulated desorption

    Slutsky, M. S.; George, T. F.

    1978-01-01

    A quantum theory of laser-stimulated desorption (LSDE) is presented and critically analyzed. It is shown how LSDE depends on laser-pulse characteristics and surface-lattice dynamics. Predictions of the theory for a Debye model of the lattice dynamics are compared to recent experimental results.

  13. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  14. Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent μ to γ1 class switch recombination in CD38-activated B cells

    Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to μ to γ1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce μ to γ1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces μ to γ1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce μ to γ1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce μ to γ1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR

  15. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  16. PAH desorption from river floodplain soils using supercritical fluid extraction

    Sequential supercritical fluid extraction (SFE) was performed in order to estimate desorption of PAHs from river floodplain soils which contain coal and coal-derived particles. Original soils, soils' light fractions (ρ -3), and <63 μm fractions were studied for PAHs' desorption kinetics. Desorption data were successfully described using a two-site model. Desorption rate constants were one order of magnitude lower than those of 'slow' and 'very slow' desorption rates from other studies. This suggests very slow and extremely slow desorption. Estimated time scales releasing 99% of total extractable contaminants ranged from decades for 2-4-ring PAHs and hundreds of years for 5-6-ring PAHs. We demonstrate that, despite high soil PAH concentrations which are due to coal and coal-derived particles, the general environmental risk is reduced by the very slow and extremely slow desorption rates. - Very slow and extremely slow PAHs desorption from carbonaceous river floodplain soils

  17. Current trends of HIV recombination worldwide

    Katherine A. Lau

    2013-06-01

    Full Text Available One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O, as well as inter- and intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs and unique recombinant forms (URFs, requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and co-circulation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.

  18. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban;

    2016-01-01

    stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious...... different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested...

  19. Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

    Mahiou, J; Abastado, J P; Cabanie, L; Godeau, F

    1998-03-01

    We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart. PMID:9480929

  20. Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

    Chung, Wen-Jen; Huang, Chi-Lung; Gong, Hong-Yi; Ou, Tsung-Yin; Hsu, Jue-Liang; Hu, Shao-Yang

    2015-06-01

    Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay. PMID:25703054

  1. Regulation of homologous recombination at telomeres in budding yeast

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that...

  2. Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

    Saharinen, Pipsa; Kerkelä, Katja; Ekman, Niklas; Marron, Marie; Brindle, Nicholas; Lee, Gyun Min; Augustin, Hellmut; Koh, Gou Young; Alitalo, Kari

    2005-01-01

    The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly ind...

  3. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

    Bandaranayake, Ashok D.; Correnti, Colin; Ryu, Byoung Y.; Brault, Michelle; Strong, Roland K.; Rawlings, David J.

    2011-01-01

    A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring

  4. Investigations for designing catalytic recombiners

    In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration

  5. Recombinant DNA in Medicine

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  6. Improving baculovirus recombination

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  7. Anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

    Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver. We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus, the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity

  8. Cell type-specific conditional regulation of the c-myc proto-oncogene by combining Cre/loxP recombination and tamoxifen-mediated activation.

    Jäger, Richard; Maurer, Jochen; Jacob, Andrea; Schorle, Hubert

    2004-03-01

    Development of inducible genetic switches for in vivo use with transgenic mice has revolutionized many areas in modern molecular biology. Combining two techniques, Cre/loxP-based genetic recombination and ligand-dependent activation of a chimeric protein, we generated transgenic mice which allow for the spatiotemporal control of expression and of activity of the proto-oncogene c-myc. To these ends, the gene encoding the tamoxifen-inducible c-mycER(T) fusion protein (mycER(T)) was inserted in the ubiquitously active ROSA 26 gene locus by gene targeting. In the resulting ROSAMER allele, generalized transcription of the mycER(T) gene is prevented by a preceding transcriptional stop sequence which is flanked by loxP sites. Crosses of ROSAMER transgenic mice with Mox2 cre transgenic mice revealed tight control of mycER(T) transcription in various tissues unless the transcriptional stop sequence was removed by cre-mediated excision. Furthermore, we were able to demonstrate tamoxifen-dependent activation of the MycER(T) protein in embryonic fibroblasts derived from such mice. As a proof of principle, we demonstrate that primary neural crest cultures established from ROSAMER mice maintain their proliferative capacity in a 4-OHT-dependent manner. Furthermore, we demonstrate that such neural crest cells retain their differentiation potential as shown by expression of NF 160, a marker of neuronal differentiation upon 4-OHT withdrawal. The transgenic mice produced may thus be valuable tools for studying the cell type-specific effects of c-myc activity in development and disease. PMID:15048812

  9. Recombinant human brain natriuretic peptide attenuates LPS-induced cellular injury in human fetal lung fibroblasts via inhibiting MAPK and NF-κB pathway activation.

    Song, Zhi; Zhao, Xiu; Liu, Martin; Jin, Hongxu; Cui, Yan; Hou, Mingxiao; Gao, Yan

    2016-08-01

    Inflammatory responses are vital in lung injury diseases, particularly acute respiratory distress syndrome (ARDS). Recombinant human brain natriuretic peptide (rhBNP) has been shown to exhibit anti‑inflammatory effects in vivo in our previous studies. The present study aimed to investigate the mechanisms underlying the anti‑inflammatory effects of rhBNP on lipopolysaccharide (LPS)-induced human fetal lung fibroblasts (HFL-1). The results showed that LPS induced a significant increase in the leakage of lactate dehydrogenase and the secretion of interleukin (IL)‑1β. Activation of p38, extracellular-signal regulated kinase (ERK) 1/2, c‑Jun NH2-terminal kinase (JNK) mitogen‑activated protein kinases (MAPK)s, and nuclear factor (NF)‑κB in HFL‑1 cells was also observed following treatment with LPS. Treatment with rhBNP (0.1 µM) reduced the production of IL‑1β at the protein and mRNA levels. Moreover, rhBNP decreased the phosphorylation of p38, ERK1/2 and JNK induced by LPS. However, the JNK inhibitor, SP600125, significantly inhibited LPS‑induced IL‑1β production. These results indicate that the inhibition of IL‑1β by may dependent upon the JNK signaling pathway. The LPS‑induced NF‑κB activation was also suppressed by rhBNP, and IL‑1β production was inhibited by the NF‑κB inhibitor. Furthermore, NF‑κB activation was attenuated by the JNK inhibitor, indicating that NF‑κB activation was dependent on the JNK signaling pathway. The present study suggests that rhBNP exhibits an anti‑inflammatory effect on LPS‑induced HFL‑1 cell injury via the inhibition of MAPK and NF‑κB signaling pathways and may exhibit therapeutic potential for acute lung injury and ARDS. PMID:27314600

  10. Enhancement of human immunodeficiency virus (HIV)-specific CD4+ and CD8+ cytotoxic T-lymphocyte activities in HIV-infected asymptomatic patients given recombinant gp160 vaccine.

    Kundu, S K; Katzenstein, D; Moses, L E; Merigan, T C

    1992-01-01

    Twenty-six human immunodeficiency virus (HIV)-infected asymptomatic patients with CD4+ lymphocytes > 400 per mm3 were randomly allocated to a range of doses of recombinant gp160 or a control (recombinant hepatitis B vaccine) on a double-blind basis. Each patient received an injection at 0, 4, 12, 24, 36, and 48 weeks. Treatment assignments were decoded when all patients reached 28 weeks of the study period. HIV-1-specific CD4+ and CD8+ cytotoxic T lymphocyte (CTL) activities were assessed in ...

  11. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein.

    Upadhyay, Arun K; Singh, Anupam; Mukherjee, K J; Panda, Amulya K

    2014-01-01

    A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form. PMID:25309524

  12. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli in to active tetrameric protein

    AmulyaKumarPanda

    2014-09-01

    Full Text Available A tetrameric protein of therapeutic importance, Escherichia coli L-Asparaginase-II was expressed in Escherichia coli as inclusion bodies. Asparaginase inclusion bodies were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps using ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from inclusion bodies was around 50 %. The melting temperature (Tm of the purified asparaginase was found to be 64 °C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 U/mg. Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the inclusion body aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of inclusion body aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. This helped in improved recovery of asparaginase in bioactive tetrameric form.

  13. Evaluation of sorption-desorption processes for metalaxyl in natural and artificial soils.

    Sukul, Premasis; Lamshöft, Marc; Zühlke, Sebastian; Spiteller, Michael

    2013-01-01

    The main process controlling soil-pesticide interaction is the sorption-desorption as influenced by active soil surfaces. The sorption phenomena can influence translocation, volatility, persistence and bioactivity of a pesticide in soil. The present investigation was conducted on natural and artificial soils in order to enumerate the effect of soil components such as montmorillonite and ferrihydrite on the sorption behaviour of the fungicide metalaxyl and if sorption-desorption of the chiral pesticide affects the enantiomeric ratio. The sorption-desorption characteristics of metalaxyl were investigated by batch equilibration technique in a natural soil, two artificial soils, and in pure montmorillonite and ferrihydrite. After extraction, pesticide residues were analyzed by conventional and chiral chromatography using tandem mass spectrometry. A KdSorp (2.3-6.5) suggests low level sorption of metalaxyl with an appreciable risk of run-off and leaching. Thus, metalaxyl poses a threat to surface and ground water contamination. Furthermore, desorption tests revealed a hysteretic effect (H ≤ 0.8) in natural and artificial soils. Significant amount of metalaxyl was found tightly bound to the adsorbents without desorbing readily after desorption cycle. Desorption of 22-56% of the total amount of the retained metalaxyl was determined. This study reveals that an artificial soil derived from different soil constituents can be used to assess their influence on sorption/desorption processes. The present investigation showed that both montmorillonite and ferrihydrite play a significant role in the sorption of metalaxyl. The sorption doesn't influence the enantiomeric ratio of racemic metalaxyl. PMID:23452208

  14. Therapeutic Recombinant Monoclonal Antibodies

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  15. Immunization with Dendritic Cells Pulsed ex vivo with Recombinant Chlamydial Protease-Like Activity Factor Induces Protective Immunity Against Genital Chlamydia muridarum Challenge

    Bernard eArulanandam

    2011-12-01

    Full Text Available We have shown that immunization with soluble recombinant (r chlamydial protease-like activity factor (rCPAF and a T helper (Th 1 type adjuvant can induce significantly enhanced bacterial clearance and protection against Chlamydia–induced pathological sequelae in the genital tract. In this study, we investigated the use of bone marrow derived dendritic cells (BMDCs pulsed ex vivo with rCPAF+CpG in an adoptive subcutaneous immunization for the ability to induce protective immunity against genital chlamydial infection. We found that BMDCs pulsed with rCPAF+CpG efficiently up-regulated the expression of activation markers CD86, CD80, CD40 and major histocompatibility complex class II (MHC II, and secreted interleukin-12, but not IL-10 and IL-4. Mice adoptively immunized with rCPAF+CpG-pulsed BMDCs or UV-EB+CpG-pulsed BMDCs produced elevated levels of antigen-specific IFN- and enhanced IgG1 and IgG2a antibodies. Moreover, mice immunized with rCPAF+CpG-pulsed BMDCs or UV-EB+CpG-pulsed BMDCs exhibited significantly reduced genital Chlamydia shedding, accelerated resolution of infection, and reduced oviduct pathology when compared to infected mock-immunized animals. These results suggest that adoptive subcutaneous immunization with ex vivo rCPAF-pulsed BMDCs is an effective approach, comparable to that induced by UV-EB-BMDCs, for inducing robust anti-Chlamydia immunity.

  16. Plasma-wall interactions data compendium-3. Hydrogen retention property, diffusion and recombination coefficients database for selected plasma-facing materials

    This report will present additional data to those included in the previous report of this series. These new data are ''effective'' hydrogen (deuterium) recombination coefficients estimated under the assumption that hydrogen recombination occurs in the bulk of materials, followed by immediate desorption to vacuum. The SI units are used for all the data collected in this report. (author)

  17. A study of the thermal desorption of deuterium from Fe-Al multilayers

    Multilayer samples have been obtained by evaporating 15 couples of 10 nm Fe and 14 nm Al layers on (100)-oriented Si wafers in an UHV chamber. After deposition the samples have been heat treated at temperatures ranging from 673 to 773 K. Some samples were also annealed in air to promote the formation of a surface oxide layer. After deposition and heat treatments the samples have been studied by Thermal Desorption Spectroscopy (TDS), Conversion Electron Moessbauer Spectroscopy (CEMS), Rutherford Backscattering Spectroscopy (RBS), Glancing Angle X-Rays Diffraction (GAXRD) and X-Ray Reflectometry (XRR) in order to achieve a complete characterisation of the deposited layers. Results can be summarised as follows: the samples are formed of the stoichiometric FeAl compound, the D2 effusion spectra showed a first peak attributed to the effusion of deuterium contained in the point defects (vacancies) produced by the ion implantation process and a second peak attributed to deuterium release from trapping sites in the FeAl lattice that could be the defect complex formed by the association of a vacancy in the Fe sublattice and a substitutional Fe atom in the Al sublattice (VFe-FeAl). The effusion kinetics can be reproduced by assuming that: (a) deuterium effusion at ∼500 K is controlled by the D2 surface recombination process with Edes = 1.57 ± 0.02 eV as activation energy; (b) deuterium effusion at ∼820 K is controlled by the release of D atoms from the trap sites, a process which occurs in connection with defects relaxation. The role of the lattice defects and the microstructure of the samples in the effusion process has been also investigated. (Authors)

  18. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  19. Matrix effects in plasma desorption mass spectrometry

    Bouchonnet, Stephane; Hoppilliard, Yannik; Mauriac, Christine

    1993-07-01

    In Plasma Desorption (PD) Mass Spectrometry, valine/matrix mixtures have been studied in order to specify the influence of a matrix during the desorption-ionization (DI) of volume. The different matrices used were carboxylic acids (barbituric acid, 2-chloronicotinic acid, 3-chloropropionic acid, cysteine, pentafluorobenzoic acid, picric acid, sinapinic acid) and CsI, an inorganic salt. Three effects are proposed to explain the influence of each matrix on the DI of valine: a physical effect, a chemical effect and a (de)cationization effect. Thermodynamic diagrams are proposed to explain each effect. Each matrix gives either a specific effect or a superimposition of effects. The concentration effect of matrices is also studied.

  20. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus–β-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The

  1. Mercury compounds characterization by thermal desorption

    Rumayor Villamil, Marta; Díaz Somoano, Mercedes; López Antón, María Antonia; Martínez Tarazona, María Rosa

    2013-01-01

    [EN] The ability to accurately determine metal mercury content and identify different mercury species in solid samples is essential for developing remediation and control strategies. The aim of the present study is to characterize mercury compounds based on thermal desorption. For this purpose a series of samples was prepared and the operational parameters—heating velocity, carrier gas—were optimized. Fifteen commercial mercury compounds were analyzed for use as fingerprints. The results of t...

  2. Comparison of recombination models in organic bulk heterojunction solar cells

    Recombination in bulk-heterojunction (BHJ) organic solar cells is the key loss mechanism, and it directly affects characteristic parameters such as power conversion efficiency, short-circuit current, open-circuit voltage, and fill factor. However, which recombination mechanism dominates the loss in organic materials is unclear at present. In this work, we simulate state-of-art BHJ solar cells using five recombination models, including direct recombination, Langevin recombination, charge transfer state recombination, trap-assisted recombination, and recombination via tail. All processes are strongly dependent on charge carrier mobility and exhibit a similar recombination distribution in active layer. For high mobilities, all models present a similar behavior along with the increased mobilities, whereas, there are slight differences in open-circuit voltage between trap/tail model and other ones at lower mobilities, resulting from the interaction between photo-carriers and dark-carriers

  3. Thermal desorption of ammonia from crystalline forsterite surfaces

    Suhasaria, T; Zacharias, H

    2015-01-01

    The thermal desorption of ammonia (NH$_3$) from single crystal forsterite (010) has been investigated using temperature-programmed desorption. The effect of defects on the desorption process has been probed by the use of a rough cut forsterite surface prepared from the cleaved forsterite sample. Several approaches have been used to extract the desorption energy and pre-exponential factor describing the desorption kinetics. In the sub-monolayer coverage regime, the NH$_3$ desorption shows a broad distribution of desorption energies, indicating the presence of different adsorption sites, which results in an apparent coverage-dependent desorption energy. This distribution is sensitive to the surface roughness with the cut forsterite surface displaying a significantly broader distribution of desorption energies compared to the cleaved forsterite surface. The cut forsterite surface exhibits sites with desorption energies up to 62.5 kJ mol$^{-1} $ in comparison to a desorption energy of up to 58.0 kJ mol$^{-1} $ fo...

  4. Recombinant microorganisms for increased production of organic acids

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  5. Light-induced atomic desorption: recent developments

    Mariotti, E.; Atutov, S. N.; Biancalana, Valerio; Bocci, S.; Burchianti, A.; Marinelli, C.; Nasyrov, K. A.; Pieragnoli, B.; Moi, L.

    2001-04-01

    Light induced atomic desorption (LIAD) is an impressive manifestation of a new class of phenomena involving alkali atoms, dielectric films and light. LIAD consists of a huge emission of alkali atoms (experimentally proved for sodium, potassium, rubidium and cesium) from siloxane films when illuminated by laser or ordinary light. Most of the experiments have been performed in glass cells suitably coated by a thin film (of the order of 10 micrometer) either of poly - (dimethylsiloxane) (PDMS), a polymer, or of octamethylcyclotetrasiloxane (OCT), a crown molecule. LIAD is a combination of two processes: direct photo-desorption from the surface and diffusion within the siloxane layer. The photo-desorbed atoms are replaced by fresh atoms diffusing to the surface. Moreover, from the experimental data it comes out that the desorbing light increases atomic diffusion and hence the diffusion coefficient. To our knowledge this is the first time that such an effect is clearly observed, measured and discussed: LIAD represents a new class of photo-effects characterized by two simultaneous phenomena due to the light: surface desorption and fastened bulk diffusion.

  6. Thermal desorption of deuterium implanted into beryllium

    Markin, A.V.; Chernikov, V.N.; Zakharov, A.P. [Institute of Physical Chemistry, Moscow (Russian Federation)] [and others

    1995-09-01

    By means of TDS measurements it is shown that the desorption of deuterium from Be implanted with 5 keV D ions to fluences, {Phi}, from 1x10{sup 20} D/m{sup 2} to 1x10{sup 21} D/m{sup 2} proceeds in one high temperature stage B, while at {Phi} {ge} 1.2x10{sup 21}D/m{sup 2} one more stage A is added. The desorption maximum A is narrow and consists of two peaks A{sub 1} and A{sub 2} at about 460 K and 490 K, respectively. Peak A{sub 1} is attributed to the desorption of deuterium from the walls of opened channels formed under D ion implantation. Peak {sub A}2 is a consequence of the opening of a part of closed bubbles/channels to the outer surface. The position of maximum B shifts noticeably and nonsteadily on the fluence in a range from 850 to 1050 K. The origin of this maximum is the liberation of D atoms bound at vacancy complexes discussed previously by Wampler. The dependence of Tm(B) on the fluence is governed by the interaction of freely migrating D atoms with partly opened or fully closed gas cavity arrangements which are created under temperature ramping, but differently in specimens implanted with D ions to different fluences.

  7. Photoionization and Recombination

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  8. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  9. Hydrogen desorption properties of magnesium hydride catalyzed multiply with carbon and silicon

    Klimkowicz, Alicja [AGH University of Science and Technology Faculty of Energy and Fuels, al. A. Mickiewicza 30, 30-059 Krakow (Poland); Shibaura Institute of Technology, Department of Engineering Science and Mechanics, 3-7-5 Toyosu, Koto-ku 135-8548, Tokyo (Japan); Takasaki, Akito, E-mail: takasaki@sic.shibaura-it.ac.jp [Shibaura Institute of Technology, Department of Engineering Science and Mechanics, 3-7-5 Toyosu, Koto-ku 135-8548, Tokyo (Japan); Gondek, Łukasz; Figiel, Henryk [AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, al. A. Mickiewicza 30, 30-059 Krakow (Poland); Świerczek, Konrad [AGH University of Science and Technology Faculty of Energy and Fuels, al. A. Mickiewicza 30, 30-059 Krakow (Poland)

    2015-10-05

    Highlights: • Crystal structure of 2 mol MgH{sub 2} + (1 − X) mol C + X mol Si (for X = 0, 0.25, 0.5, 0.75 and 1). • Enhanced hydrogen desorption properties. • Lowered temperature of hydrogen desorption for MgH{sub 2} with the multiple addition of C and Si. • Distribution of Si and C on MgH{sub 2} particles after mechanical milling. - Abstract: Magnesium hydride (MgH{sub 2}) is considered as one of hydrogen storage materials. However, the application is limited because of slow hydrogen kinetics and high thermodynamic stability. In this study, MgH{sub 2} powders were mechanically milled with graphite (C) and/or silicon (Si) powders, and effect of multiple addition of C and Si on hydrogen desorption properties was investigated. The multiple addition caused a decrease of the hydrogen desorption (onset and peak) temperatures more than single addition, and the lowest activation energy for hydrogen desorption, 62 kJ/mol, was obtained from sample powders of (2 mol MgH{sub 2} + 0.5 mol C + 0.5 mol Si). The surface of the sample powders after mechanical milling revealed a good distribution of C on the MgH{sub 2} surface and a presence of finer Si particles.

  10. Hydrogen desorption properties of magnesium hydride catalyzed multiply with carbon and silicon

    Highlights: • Crystal structure of 2 mol MgH2 + (1 − X) mol C + X mol Si (for X = 0, 0.25, 0.5, 0.75 and 1). • Enhanced hydrogen desorption properties. • Lowered temperature of hydrogen desorption for MgH2 with the multiple addition of C and Si. • Distribution of Si and C on MgH2 particles after mechanical milling. - Abstract: Magnesium hydride (MgH2) is considered as one of hydrogen storage materials. However, the application is limited because of slow hydrogen kinetics and high thermodynamic stability. In this study, MgH2 powders were mechanically milled with graphite (C) and/or silicon (Si) powders, and effect of multiple addition of C and Si on hydrogen desorption properties was investigated. The multiple addition caused a decrease of the hydrogen desorption (onset and peak) temperatures more than single addition, and the lowest activation energy for hydrogen desorption, 62 kJ/mol, was obtained from sample powders of (2 mol MgH2 + 0.5 mol C + 0.5 mol Si). The surface of the sample powders after mechanical milling revealed a good distribution of C on the MgH2 surface and a presence of finer Si particles

  11. Reversed DNA Strand Cleavage Specificity in Initiation of Cre–LoxP Recombination Induced by the His289Ala Active-site Substitution

    Gelato, Kathy A.; Martin, Shelley S.; Baldwin, Enoch P.

    2005-01-01

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set...

  12. Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

    Huang, Qiongying; Gershenson, Anne; Roberts, Mary F

    2016-06-01

    The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of L. monocytogenes. PMID:26976751

  13. Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV.

    Vamvaka, Evangelia; Arcalis, Elsa; Ramessar, Koreen; Evans, Abbey; O'Keefe, Barry R; Shattock, Robin J; Medina, Vicente; Stöger, Eva; Christou, Paul; Capell, Teresa

    2016-06-01

    Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS) GRFT in the best-performing plants was 223 μg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. (OS) GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified (OS) GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS) GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component. PMID:26800650

  14. Overproduction and biological activity of prodigiosin-like pigments from recombinant fusant of endophytic marine Streptomyces species.

    El-Bondkly, Ahmed M A; El-Gendy, Mervat M A; Bassyouni, Rasha H

    2012-11-01

    Thirty-four endophytic marine Actinomycetes isolates were recovered from the Egyptian marine sponge Latrunculia corticata, out of them 5 isolates (14.7 %) showed red single colonies on yeast-CzAPEK plates. Isolates under the isolation code NRC50 and NRC51 were observed with the strongest red biomass. After application of protoplast fusion between NRC50 and NRC51 isolates, 26 fusants were selected and produced widely different amounts of prodigiosin-like pigments (PLPs) on different fermentation media. Among them fusant NRCF69 produced 79 and 160.4 % PLPs more than parental strains NRC50 and NRC51, respectively. According to the analysis of 16S rDNA sequence (amplified, sequenced, and submitted to GenBank under Accession no. JN232405 and JN232406, respectively), together with their morphological and biochemical characteristics, parental strains NRC50 (P1) and NRC51 (P2) were identified as Streptomyces sp. and designated as Streptomyces sp. NRC50 and Streptomyces sp. NRC51. This study describes a low cost, effective production media by using peanut seed broth, sunflower oil broth or dairy processing wastewater broth alone, or supplemented with 0.5 % mannitol that supports the production of PLPs by the Streptomyces fusant NRCF69 under study (42.03, 40.11, 36.7 and 47 g L(-1), respectively). PLPs compounds exhibited significant cytotoxic activities against three human cancer cell lines: colon cancer cell line (HCT-116), liver cancer cell line (HEPG-2) and breast cancer cell line (MCF-7) and antimycotic activity against clinical dermatophyte isolates of Trichophyton, Microsporum and Epidermophyton. PMID:22777253

  15. Recombineering Homologous Recombination Constructs in Drosophila

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A.; Williams, Nathan David; Hiesinger, P. Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineeri...

  16. Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

    XIANG-MING LI; FANG-NI LUO; GuI-XIA LIU; PING-TING ZHU

    2008-01-01

    Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on ndustrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section (influents) and 9 from post-treatment section (effluents), were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 1713-estradiol (E2), estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

  17. In vivo distribution of recombinant interleukin-2-activated autologous lymphocytes administered by intra-arterial infusion in patients with renal cell carcinoma

    Recombinant interleukin-2 (RIL 2)-activated autologous peripheral blood lymphocytes (PBL) were infused directly into the renal arteries of 3 patients with renal cell carcinoma, and the in vivo distribution of the infused cells was investigated. In vitro studies to define the optimal culture conditions indicated that maximal lymphokine-activated killer activity was observed at around 10-20 days in culture, as judged by the cytotoxicity against fresh allogenic tumor cells. Maximal expression of the interleukin-2 receptor was also obtained at around 10 days. PBL collected by leukopheresis from each patient were thus cultured for 10 days with RIL 2, labeled with 111In-oxine, and then infused directly into the renal artery of the affected kidney via a catheter. Radioactivity in the infused side of the kidneys increased immediately after the infusion but then gradually decreased. Radioactivity in the lungs also rapidly increased within the first hour but then cleared gradually, whereas that in the liver and spleen tended to increase steadily. Nevertheless, at 48 hours, the infused side of the kidneys retained levels of radioactivity comparable to those seen in the liver and spleen, while the levels seen in the lungs were already close to background levels. The radioactivity in the areas corresponding to tumors remained consistently higher than that in the normal parts of the affected kidneys. The direct comparison of the radioactivity distribution pattern with the macroscopic appearance of surgically resected kidneys indicated that the accumulation of radioactivity was indeed selectively associated with the tumor tissues in the kidneys, except for a case in which the tumor was quite necrotic and hypovascular

  18. WATER ADSORPTION AND DESORPTION ISOTHERMS ON MILK POWDER: II. WHOLE MILK

    Edgar M. Soteras

    2014-03-01

    Full Text Available The aim of this research was the determination of adsorption and desorption isotherms of cow whole milk powder. The experiments have been carried out at 15, 25 and 40 ºC, in ranges of moisture and water activity characteristic of normal conditions in which the processes of drying, packaging and storage are developed. By studying the influence of the temperature on the experimental plots, the isosteric adsorption heat was determined. Experimental data were correlated to the referential model of Guggenheim, Anderson and Boer (GAB. For both, adsorption and desorption, a good model fit was observed. The isotherms showed very similar shapes between them and, by comparing adsorption and desorption isotherms, the phenomenon of hysteresis was confirmed.

  19. Non-isothermal kinetics of the thermal desorption of mercury from a contaminated soil

    The Almaden mining district (Ciudad Real, Spain) was the largest cinnabar (mercury sulphide) mine in the world. Its soils have high levels of mercury a consequence of its natural lithology, but often made much worse by its mining history. The present work examines the thermal desorption of two contaminated soils from the Almaden area under non-isothermal conditions in a N2 atmosphere, using differential scanning calorimetry (DSC). DSC was performed at different heating rates between room temperature and 600 degree centigrade. Desorption temperatures for different mercury species were determined. The Friedman, Flynn-Wall-Ozawa and Coasts Redfern methods were employed to determine the reaction kinetics from the DSC data. The activation energy and pre-exponential factor for mercury desorption were calculated. (Author)

  20. Antitumor activity of recombinant Bacille Calmette-Guérin secreting interleukin-15-Ag85B fusion protein against bladder cancer.

    Takeuchi, Ario; Eto, Masatoshi; Tatsugami, Katsunori; Shiota, Masaki; Yamada, Hisakata; Kamiryo, Yoriyuki; Dejima, Takashi; Kashiwagi, Eiji; Kiyoshima, Keijiro; Inokuchi, Junichi; Takahashi, Ryosuke; Yokomizo, Akira; Ohara, Naoya; Yoshikai, Yasunobu

    2016-06-01

    Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is used for the treatment of bladder cancer. The recruitment of neutrophlis to the bladder after BCG instillation exerts anti-tumor activity against bladder tumor. We have recently demonstrated that interleukin (IL)-17A produced by γδ T cells played a role in the recruitment of neutrophlis to the bladder after BCG instillation. IL-15 is known to play an important role in neutrophil migration during inflammation. We previously constructed a recombinant BCG strain expressing the fusion protein of IL-15 and Ag85B (BCG-IL-15) for prevention of Mycobacterium tuberculosis infection. Here we compared the efficacy of the BCG-IL-15 in protection against bladder cancer with that of rBCG-Ag85B (BCG). Six-week-old female C57BL/6 mice were inoculated with MB49 bladder tumor cells in the bladder and subsequently intravesically inoculated with BCG or BCG-IL-15. BCG-IL-15 treatment significantly prolonged survival of mice inoculated with bladder cancer cells compared with BCG treatment. Infiltration of neutrophils was significantly elevated in BCGB-IL-15 treated mice accompanied by increased chemokines (MIP-2 and MIP-1α) in the bladder. Thus, BCG-IL-15 exerted additive effect on Infiltration of neutrophils in the bladder. BCG-IL-15 may be a promising drug for non-muscle invasive bladder cancer. PMID:27093372

  1. A Shark Liver Gene-Derived Active Peptide Expressed in the Silkworm, Bombyx mori: Preliminary Studies for Oral Administration of the Recombinant Protein

    Jun Li

    2013-05-01

    Full Text Available Active peptide from shark liver (APSL is a cytokine from Chiloscyllium plagiosum that can stimulate liver regeneration and protects the pancreas. To study the effect of orally administered recombinant APSL (rAPSL on an animal model of type 2 diabetes mellitus, the APSL gene was cloned, and APSL was expressed in Bombyx mori N cells (BmN cells, silkworm larvae and silkworm pupae using the silkworm baculovirus expression vector system (BEVS. It was demonstrated that rAPSL was able to significantly reduce the blood glucose level in mice with type 2 diabetes induced by streptozotocin. The analysis of paraffin sections of mouse pancreatic tissues revealed that rAPSL could effectively protect mouse islets from streptozotocin-induced lesions. Compared with the powder prepared from normal silkworm pupae, the powder prepared from pupae expressing rAPSL exhibited greater protective effects, and these results suggest that rAPSL has potential uses as an oral drug for the treatment of diabetes mellitus in the future.

  2. The immobilization of recombinant human tropoelastin on metals using a plasma-activated coating to improve the biocompatibility of coronary stents.

    Waterhouse, Anna; Yin, Yongbai; Wise, Steven G; Bax, Daniel V; McKenzie, David R; Bilek, Marcela M M; Weiss, Anthony S; Ng, Martin K C

    2010-11-01

    Current endovascular stents have sub-optimal biocompatibility reducing their clinical efficacy. We previously demonstrated a plasma-activated coating (PAC) that covalently bound recombinant human tropoelastin (TE), a major regulator of vascular cells in vivo, to enhance endothelial cell interactions. We sought to develop this coating to enhance its mechanical properties and hemocompatibility for application onto coronary stents. The plasma vapor composition was altered by incorporating argon, nitrogen, hydrogen or oxygen to modulate coating properties. Coatings were characterized for 1) surface properties, 2) mechanical durability, 3) covalent protein binding, 4) endothelial cell interactions and 5) thrombogenicity. The N(2)/Ar PAC had optimal mechanical properties and did not delaminate after stent expansion. The N(2)/Ar PAC was mildly hydrophilic and covalently bound the highest proportion of TE, which enhanced endothelial cell proliferation. Acute thrombogenicity was assessed in a modified Chandler loop using human blood. Strikingly, the N(2)/Ar PAC alone reduced thrombus weight by ten-fold compared to 316L SS, a finding unaltered with immobilized TE. Serum soluble P-selectin was reduced on N(2)/Ar PAC and N(2)/Ar PAC + TE (p coating for metal alloys with multifaceted biocompatibility that resists delamination and is non-thrombogenic, with implications for improving coronary stent efficacy. PMID:20708259

  3. Safety and efficacy of recombinant activated factor VII: a randomized placebo-controlled trial in the setting of bleeding after cardiac surgery

    Gill, Ravi; Herbertson, Mike; Vuylsteke, Alain;

    2009-01-01

    BACKGROUND: Blood loss is a common complication of cardiac surgery. Evidence suggests that recombinant activated factor VII (rFVIIa) can decrease intractable bleeding in patients after cardiac surgery. Our objective was to investigate the safety and possible benefits of rFVIIa in patients who bleed...... after cardiac surgery. METHODS AND RESULTS: In this phase II dose-escalation study, patients who had undergone cardiac surgery and were bleeding were randomized to receive placebo (n=68), 40 microg/kg rFVIIa (n=35), or 80 microg/kg rFVIIa (n=69). The primary end points were the number of patients....../kg, 14%; P=0.25; 80 microg/kg, 12%; P=0.43). After randomization, significantly fewer patients in the rFVIIa group underwent a reoperation as a result of bleeding (P=0.03) or required allogeneic transfusions (P=0.01). CONCLUSIONS: On the basis of this preliminary evidence, rFVIIa may be beneficial for...

  4. A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model

    S Roy

    2015-01-01

    Full Text Available Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 10 4.25 LD 50 /0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD 50 titre, challenge dose of 50 LD 50 was administered along with rhuIFN α-2a with pre-exposure (primed and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These findings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the efficacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease.

  5. A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model.

    Roy, S; Patil, D; Ghadigaonkar, S; Roy, R; Mukherjee, S; Chowdhary, A; Deshmukh, R

    2015-01-01

    Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 10(4.25) LD50/0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD50 titre, challenge dose of 50 LD 50 was administered along with rhuIFN α-2a with pre-exposure (primed) and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These findings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the efficacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease. PMID:25560017

  6. Solvent-induced desorption of alkanethiol ligands from Au nanoparticles.

    Huang, Yuanyuan; Liu, Wei; Cheng, Hao; Yao, Tao; Yang, Lina; Bao, Jie; Huang, Ting; Sun, Zhihu; Jiang, Yong; Wei, Shiqiang

    2016-06-21

    Removing surfactants from a colloidal metal nanoparticle surface is necessary for their realistic applications, and how they could be stripped is a subject of active investigation. Here, we report a solvent-induced desorption of dodecanethiol ligands from the gold nanoparticle surface, and traced this desorption process using a combination of in situ X-ray absorption fine structure (XAFS) and Raman spectroscopic techniques. In situ analysis results reveal that the solvent exchange of ethanol with tetrahydrofuran (THF) can effectively remove dodecanethiol ligands while keeping the particle morphology unchanged. Upon increasing the THF/ethanol ratio from 0 : 1 to 5 : 1, the surface coverage of thiol on the Au surface is reduced from 0.47 to 0.07, suggesting the depletion of ligands first from the nanoparticle facet sites, then from the edge sites, while the ligands at the corner sites are intact. This work enriches our knowledge on surfactant removal and may pave the way towards preparing surface-clean nanoparticles for practical applications. PMID:27241025

  7. Optical detection of CO and CO2 temperature dependent desorption from carbon nanotube clusters

    The development of new materials relies on high precision methods to quantify adsorption/desorption of gases from surfaces. One commonly used approach is temperature programmed desorption spectroscopy. While this approach is very accurate, it requires complex instrumentation, and it is limited to performing experiments under high vacuum, thus restricting experimental scope. An alternative approach is to integrate the surface of interest directly onto a detector face, creating an active substrate. One surface that has applications in numerous areas is the carbon nanotube (CNT). As such, an active substrate that integrates a CNT surface on a sensor and is able to perform measurements in ambient environments will have significant impact. In the present work, we have developed an active substrate that combines an optical sensor with a CNT cluster substrate. The optical sensor is able to accurately probe the temperature dependent desorption of carbon monoxide and carbon dioxide gases from the CNT cluster surface. This active substrate will enable a wide range of temperature dependent desorption measurements to be performed from a scientifically interesting material system. (paper)

  8. Desorption electrospray ionization with a portable mass spectrometer: in situ analysis of ambient surfaces.

    Mulligan, Christopher C; Talaty, Nari; Cooks, R Graham

    2006-04-28

    Desorption electrospray ionization (DESI) is implemented on a portable mass spectrometer and used to demonstrate in situ detection of active ingredients in pharmaceutical preparations, alkaloids in plant tissues, explosives, chemical warfare agent simulants and agricultural chemicals from a variety of surfaces; air monitoring applications using DESI are also introduced. PMID:16609779

  9. Direct pathway for sticking/desorption of H2 on Si(100)

    Kratzer, Peter; Hammer, Bjørk; Nørskov, Jens Kehlet

    1995-01-01

    The energetics of H2 interacting with the Si(100) surface is studied by means of ab initio total-energy calculations within the framework of density-functional theory. We find a direct desorption pathway from the monohydride phase that is compatible with experimental activation energies and...

  10. Fundamental study of recombination and recombineering in Escherichia coli

    Sun, Xiaohang; Huang, Yang

    2008-01-01

    Recombination and recombineering systems have been used in Escherichia coli to recombinant DNA sequences. With endonuclease and DNA lipase the bacterial plasmid and target DNA fragment can bind together and recombinant for a new DNA sequences. Red Proteins have been used in recombineering system to perform the function as the enzymes in recombination system, and faster and easier than the other way of recombinant new DNA sequences in E.coli. In this report we get to know the pr...

  11. Current-Driven Hydrogen Desorption from Graphene: Experiment and Theory.

    Gao, Li; Pal, Partha Pratim; Seideman, Tamar; Guisinger, Nathan P; Guest, Jeffrey R

    2016-02-01

    Electron-stimulated desorption of hydrogen from the graphene/SiC(0001) surface at room temperature was investigated with ultrahigh vacuum scanning tunneling microscopy and ab initio calculations in order to elucidate the desorption mechanisms and pathways. Two different desorption processes were observed. In the high electron energy regime (4-8 eV), the desorption yield is independent of both voltage and current, which is attributed to the direct electronic excitation of the C-H bond. In the low electron energy regime (2-4 eV), however, the desorption yield exhibits a threshold dependence on voltage, which is explained by the vibrational excitation of the C-H bond via transient ionization induced by inelastic tunneling electrons. The observed current independence of the desorption yield suggests that the vibrational excitation is a single-electron process. We also observed that the curvature of graphene dramatically affects hydrogen desorption. Desorption from concave regions was measured to be much more probable than desorption from convex regions in the low electron energy regime (∼2 eV), as would be expected from the identified desorption mechanism. PMID:26787160

  12. Current-Driven Hydrogen Desorption from Graphene: Experiment and Theory

    Gao, L.; Pal, Partha P.; Seideman, Tamar; Guisinger, Nathan P.; Guest, Jeffrey R.

    2016-02-04

    Electron-stimulated desorption of hydrogen from the graphene/SiC(0001) surface at room temperature was investigated with ultrahigh vacuum scanning tunneling microscopy and ab initio calculations in order to elucidate the desorption mechanisms and pathways. Two different desorption processes were observed. In the high electron energy regime (4-8 eV), the desorption yield is independent of both voltage and current, which is attributed to the direct electronic excitation of the C-H bond. In the low electron energy regime (2-4 eV), however, the desorption yield exhibits a threshold dependence on voltage, which is explained by the vibrational excitation of the C-H bond via transient ionization induced by inelastic tunneling electrons. The observed current-independence of the desorption yield suggests that the vibrational excitation is a singleelectron process. We also observed that the curvature of graphene dramatically affects hydrogen desorption. Desorption from concave regions was measured to be much more probable than desorption from convex regions in the low electron energy regime (~ 2 eV), as would be expected from the identified desorption mechanism

  13. Identification, cloning and lactonase activity of recombinant protein of N-acyl homoserine lactonase (AiiA from Bacillus thuringiensis 147-115-16 strain.

    Alvaro Mauricio Florez Escobar

    2014-06-01

    Full Text Available Título en español: Identificación, clonación y actividad lactonasa de la proteína recombinante de N-ácil homoserina lactonasa (AiiA de Bacillus thuringiensis cepa 147-115-16 Short title: N-acyl homoserine lactonase (AiiA from Bacillus thuringiensis Abstract: The quorum-quenching N-acyl homoserine lactonases are a family of bacterial metalloenzymes that participate in degradation of N-acyl homoserine lactones (AHLs, disrupting the quorum sensing system of gram negative bacterial species. From a collection of Bacillus thuringiensis strains isolated in Colombia from plants and exhibiting toxic activity against lepidopteran insects, 310 bacterial isolates were tested to determine lactonase activity by using biosensor systems in presence of synthetic N-hexanoyl-L-homoserine lactone (C6-HSL and N-octanoyl-L-homoserine lactone (C8-HSL. From them, 251 strains showed degrading activity to both C6-HSL and C8-HSL, 57% exhibited degrading activity to C6-HSL and 43% to C8-HSL. One B. thuringiensis strain, denoted as 147-115-16, that exhibit high degrading activity to C6-HSL and C8-HSL, was able to attenuate soft rot symptoms in infected potato slices with Pectobacterium carotovorum. This strain contains an homologous of the aiiA gene that was cloned, sequenced and expressed in Esherichia coli DE3. The recombinant protein AiiA147-11516 displays activity to C6-HSL, C8-HSL, N-(β-ketocaproyl (3-O-C6-HSL and N-3-oxo-dodecanoyl (3-O-C12-HSL. The recombinant strain in the presence of P. caratovorum cultures was able to attenuate the infection, suggesting that it interferes either with the accumulation or with the response to the AHLs signals. Acording to this data and based on previous report from recombinant AiiA147-11516, this enzyme exhibits activity to a wide range of catalytic substrates suggesting its industrial application in the disease control programs through plants transformation.Key words: lactones, Quorum sensing, Quorum quenching, Lactonases

  14. CONSTRUCTION OF ACTIVE RECOMBINANT CASPASE-3 EUKARYOTIC EXPRESSION PLA SMID AND EFFECT OF r-CASPASE-3 ON APOPTOSIS OF PANCREATIC CARCINOMA CELLS

    2001-01-01

    Objective To construct active recombinant cas pa ses-3 gene(r-caspases-3)eukaryotic expression plasmid and observe the apoptos is inducing activity of r-caspase-3 in pancreatic carcinoma cells. Methods pcDNA3.1(+)/r-caspase-3 was constructed and pan creatic carcinoma cells(PC-Ⅱ)were transfected with the pcDNA3.1(+)/r-caspases -3 by liposomes(LipofectAMINE).The expression of r-Caspase-3 mRNA in pancreat ic carcinoma cells was detected by reverse transcription process of the polymera se chain reaction(RT-PCR), and the signs of apoptosis were examined in pancreat ic carcinoma cells by the methods of the DNA electrophoresis and flow cytometry analysis(FACS).Results The sequence inserted in pBlueSKM/r-Caspase-3 p lasmid was coincident with that of the r-caspases-3. The evaluation result of pcDNA3.1(+)/r-caspases-3 through enzyme cutting was correct. A 894bp strap was observed by RT-PCR after pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/r-caspases-3 by liposomes. No strap was found in control groups. A characteristic DNA ladder was observed in pancreatic carcinoma cells DNA elect r ophoresis, and transparent hypodiploid karyotype peak was found by FACS. Conclusion The plasmid of pcDNA3.1(+)/r-Caspase-3 was c onstructed successfully, the expression of r-Caspase-3 mRMA in pancreatic carc inoma cells was confirmed by RT-PCR, and pcDNA3.1(+)/r-Caspase-3 can induce a poptosis in pancreatic carcinoma cells.

  15. An Approach to Breast Cancer Immunotherapy: The Apoptotic Activity of Recombinant Anti-Interleukin-6 Monoclonal Antibodies in Intact Tumour Microenvironment of Breast Carcinoma.

    Abou-Shousha, S; Moaaz, M; Sheta, M; Motawea, M A

    2016-06-01

    Current work is one of our comprehensive preclinical studies, a new approach to breast cancer (BC) immunotherapy through induction of tumour cell apoptosis. Tumour growth is not just a result of uncontrolled cell proliferation but also of reduced apoptosis. High levels of interleukin-6 (IL-6) are associated with metastatic BC and correlated with poor survival as it promotes growth of tumour-initiating cells during early tumorigenesis protecting these cells from apoptosis. Therefore, this study aims at investigating the potential of anti-IL-6 monoclonal antibodies to suppress IL-6 proliferative/anti-apoptotic activities in intact tumour microenvironment of BC. Fresh sterile tumour and normal breast tissue specimens were taken from 50 female Egyptian patients with BC undergoing radical mastectomy. A unique tissue culture system designed to provide cells of each intact tumour/normal tissue sample with its proper microenvironment either supplemented or not with anti-IL-6 monoclonal antibodies. To evaluate the apoptotic activity of anti-IL-6 as a novel candidate for BC treatment strategy, we compared its effects with those obtained using tumour necrosis-related apoptosis-inducing ligand TRAIL as an established apoptotic agent. Our results revealed that levels of either anti-IL-6- or TRAIL-induced apoptosis in the tumour or normal tissue cultures were significantly higher than those in their corresponding untreated ones (P Recombinant anti-IL-6 monoclonal antibodies could represent a novel effective element of immunotherapeutic treatment strategy for BC. The selectivity and anti-apoptotic potential of anti-IL-6 is highly hopeful in IL-6- abundant BC tumour microenvironment. PMID:26971879

  16. Recombinant Tissue Plasminogen Activator Induces Neurological Side Effects Independent on Thrombolysis in Mechanical Animal Models of Focal Cerebral Infarction: A Systematic Review and Meta-Analysis.

    Mei-Xue Dong

    Full Text Available Recombinant tissue plasminogen activator (rtPA is the only effective drug approved by US FDA to treat ischemic stroke, and it contains pleiotropic effects besides thrombolysis. We performed a meta-analysis to clarify effect of tissue plasminogen activator (tPA on cerebral infarction besides its thrombolysis property in mechanical animal stroke.Relevant studies were identified by two reviewers after searching online databases, including Pubmed, Embase, and ScienceDirect, from 1979 to 2016. We identified 6, 65, 17, 12, 16, 12 and 13 comparisons reporting effect of endogenous tPA on infarction volume and effects of rtPA on infarction volume, blood-brain barrier, brain edema, intracerebral hemorrhage, neurological function and mortality rate in all 47 included studies. Standardized mean differences for continuous measures and risk ratio for dichotomous measures were calculated to assess the effects of endogenous tPA and rtPA on cerebral infarction in animals. The quality of included studies was assessed using the Stroke Therapy Academic Industry Roundtable score. Subgroup analysis, meta-regression and sensitivity analysis were performed to explore sources of heterogeneity. Funnel plot, Trim and Fill method and Egger's test were obtained to detect publication bias.We found that both endogenous tPA and rtPA had not enlarged infarction volume, or deteriorated neurological function. However, rtPA would disrupt blood-brain barrier, aggravate brain edema, induce intracerebral hemorrhage and increase mortality rate.This meta-analysis reveals rtPA can lead to neurological side effects besides thrombolysis in mechanical animal stroke, which may account for clinical exacerbation for stroke patients that do not achieve vascular recanalization with rtPA.

  17. Experimental study on effects of CBM temperature-rising desorption

    MA Dong-min; LIN Ya-bing

    2012-01-01

    To study the effects of CBM (coal bed methane) temperature-rising desorption,isothermal adsorption/desorption experiments on three ranks (anthracite,coking coal and lignite) of coal at different temperatures were designed based on the traditional CBM decompression desorption.The experimental results indicate that temperature-rising desorption is more effective in high-rank coal,and ever-increasing temperature of high-rank coal reservoir can reduce the negative effects of coal matrix shrinkage in the process of production and improve the permeability of the coal reservoir as well.It is also revealed that the technique of temperature-rising desorption applied in higher-rank coal reservoir can enhance CBM recovery ratio.This study provided theoretical support for the application of temperature-rising desorption technique in practical discharging and mining projects,which can effectively tackle the gas production bottleneck problem.

  18. The effect of selective desorption mechanisms during interstellar ice formation

    Kalvans, Juris

    2015-01-01

    Major components of ices on interstellar grains in molecular clouds - water and carbon oxides - occur at various optical depths. This implies that selective desorption mechanisms are at work. An astrochemical model of a contracting low-mass molecular cloud core is presented. Ice was treated as consisting of the surface and three subsurface layers (sublayers). Photodesorption, reactive desorption, and indirect reactive desorption were investigated. The latter manifests itself through desorption from H+H reaction on grains. Desorption of shallow subsurface species was included. Modeling results suggest the existence of a "photon-dominated ice" during the early phases of core contraction. Subsurface ice is chemically processed by interstellar photons, which produces complex organic molecules. Desorption from the subsurface layer results in high COM gas-phase abundances at Av = 2.4...10mag. This may contribute towards an explanation for COM observations in dark cores. It was found that photodesorption mostly gove...

  19. Displacement desorption test of coalbed methane and its mechanism exploring

    ZHANG Suian; HUO Yongzhong; YE Jianping; TANG Shuheng; MA Dongmin

    2005-01-01

    Through the test of CH4 displaced by CO2 using the coal sample as the adsorbent, this paper has found the coalbed methane (CBM) displacement desorption phenomenon under the natural conditions and CBM mining conditions. With the help of the adsorption theory of the modern physical chemistry and interfacial chemistry, the CBM competitive adsorption and displacement desorption mechanism are intensively discussed, and a new path for studying the CBM desorption mechanism in the CBM exploitation process is explored.

  20. Effects of Lateral Diffusion on the Dynamics of Desorption

    Juwono, Tjipto; Hamad, Ibrahim Abou; Rikvold, Per Arne

    2012-01-01

    The adsorbate dynamics during simultaneous action of desorption and lateral adsorbate diffusion is studied in a simple lattice-gas model by kinetic Monte Carlo simulations. It is found that the action of the coverage-conserving diffusion process during the course of the desorption has two distinct, competing effects: a general acceleration of the desorption process, and a coarsening of the adsorbate configuration through Ostwald ripening. The balance between these two effects is governed by t...

  1. Analgesic drug delivery via recombinant tissue plasminogen activator and microRNA-183-triggered opening of the blood-nerve barrier.

    Yang, Shaobing; Krug, Susanne M; Heitmann, Johanna; Hu, Liu; Reinhold, Ann Kristin; Sauer, Solange; Bosten, Judith; Sommer, Claudia; Fromm, Michael; Brack, Alexander; Rittner, Heike L

    2016-03-01

    The peripheral nerve contains three barriers which include the blood-nerve barrier consisting of endoneurial vessels and the perineurium as well as autotypic junctions in Schwann cells. The perineurium prevents diffusion of perineurally injected drugs that can be used for selective regional pain control. It is composed of a basal membrane and layers of perineurial cells sealed by tight junction proteins like claudin-1. Claudin-1 expression and barrier function are regulated via low-density lipoprotein receptor-related protein (LRP-1). Perisciatic application of recombinant tissue plasminogen activator (rtPA) or the catalytically inactive rtPAi - both agonists of LRP-1 - reduced claudin-1 mRNA and protein expression in the rat nerve. This facilitated an increase of nociceptive thresholds after local application of hydrophilic opioids or the voltage gated sodium channel blocker (NaV1.7) ProToxin-II without apparent nerve toxicity. RtPA-induced barrier opening was mediated by LRP-1 and intracellularly by Erk phosphorylation. In silico, microRNA (miR)-rno-29b-2-5p and rno-miR-183-5p were identified as potential regulators of claudin-1 transcription in the rat. RtPA application increased miR-183-5p in the sciatic nerve. MiR-183-5p mimics functionally opened the perineurium and downregulated claudin-1 expression in vivo. In vitro, hsa-miR-183-3p mimics reduced claudin-1 expression in human HT-29/B6 cells. Overall, rtPA regulates perineurial barrier tightness via LRP-1, Erk phosphorylation and miR-183-5p/3p. This mechanism might serve as a new principle to facilitate drug delivery to peripheral nerves in humans. PMID:26735170

  2. Effects of Lateral Diffusion on the Dynamics of Desorption

    Juwono, Tjipto; Rikvold, Per Arne

    2012-01-01

    The adsorbate dynamics during simultaneous action of desorption and lateral adsorbate diffusion is studied in a simple lattice-gas model by kinetic Monte Carlo simulations. It is found that the action of the coverage-conserving diffusion process during the course of the desorption has two distinct, competing effects: a general acceleration of the desorption process, and a coarsening of the adsorbate configuration through Ostwald ripening. The balance between these two effects is governed by the structure of the adsorbate layer at the beginning of the desorption process.

  3. Substrate-Enhanced Micro Laser Desorption Ionization Mass Spectrometry Project

    National Aeronautics and Space Administration — Aerodyne Research, Inc. and the University of Massachusetts at Amherst will collaborate to develop laser desorption ionization (LDI) mass spectrometric analysis of...

  4. Testosterone sorption and desorption: Effects of soil particle size

    Qi, Yong, E-mail: yqi01@unomaha.edu [Civil Engineering Dept., University of Nebraska-Lincoln at Omaha Campus, Omaha, NE 68182 (United States); Zhang, Tian C. [Civil Engineering Dept., University of Nebraska-Lincoln at Omaha Campus, Omaha, NE 68182 (United States); Ren, Yongzheng [School of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074 (China)

    2014-08-30

    Graphical abstract: - Highlights: • Smaller soil particles have higher sorption and lower desorption rates. • The sorption capacity ranks as clay > silt > sand. • Small particles like clays have less potential for desorption. • Colloids (clays) have high potential to facilitate the transport of hormones in soil–water environments. - Abstract: Soils contain a wide range of particles of different diameters with different mobility during rainfall events. Effects of soil particles on sorption and desorption behaviors of steroid hormones have not been investigated. In this study, wet sieve washing and repeated sedimentation methods were used to fractionate the soils into five ranges. The sorption and desorption properties and related mechanisms of testosterone in batch reactors filled with fractionated soil particles were evaluated. Results of sorption and desorption kinetics indicate that small soil particles have higher sorption and lower desorption rates than that of big ones. Thermodynamic results show the sorption processes are spontaneous and exothermal. The sorption capacity ranks as clay > silt > sand, depending mainly on specific surface area and surface functional groups. The urea control test shows that hydrogen bonding contributes to testosterone sorption onto clay and silt but not on sand. Desorption tests indicate sorption is 36–65% irreversible from clay to sand. Clays have highest desorption hysteresis among these five soil fractions, indicating small particles like clays have less potential for desorption. The results provide indirect evidence on the colloid (clay)-facilitated transport of hormones (micro-pollutants) in soil environments.

  5. Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds.

    Pierce, G F; Tarpley, J. E.; Tseng, J; Bready, J; Chang, D.; Kenney, W C; Rudolph, R; Robson, M. C.; Vande Berg, J.; Reid, P.

    1995-01-01

    Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We d...

  6. Desorption of trihalomethanes in gas liquid contactors

    Updated studies show that gastric cancer is related with the existence of trihalomethanes (THMs) in the drinking water. The trihalomethanes are sub products from the degradation of humic acids and your reaction with chlorine and bromine used like decontaminates. The desorption process is used to eliminate the THMs with air in contact with the water. The experimental design was used in three contactors. The contactors selected were: the bubbling's column, the packed column and the shaken tank without screen. There were selected three variable: initial concentration of THMs, the residence time and the turbulence degree (measured with the Reynolds number). The concentrations were made with a gas chromatograph. The objective of this project is to do a comparison with the gas liquid contactors more used in the industrial level to determinate which ones are the best in the desorption process. The conclusion of the experimental design is that the tank is the equipment with the best capacity to eliminate THMs. Too it includes other techniques to eliminate THMs of the water and your treatment

  7. Plasma desorption mass spectrometry of biomolecules

    In the present work the positive and negative ion mass spectrum of nine xenobiotica metabolites conjugated with N-acetyl-cysteine (=mercapturic acids) were investigated. Whereas in the positive ion spectra most fragment ions are correlated to the backbone of the mercapturic acids the negative ion spectra were dominated by the ionized side chain. Further the peptidoglycans of the cell walls of the cyanelles of Cyanophora paradoxa of Escherichia coli were characterized by high performance liquid chromatography/plasma desorption mass spectroscopy off line. Some contributions were also done for the elucidation of suppression effects in the mass spectrometric mixture analysis of peptides. These suppression effects were correlated to peptide hydrophobicity / hydrophilicity, peptide net charge and peptide gas phase basicity. A very interesting contribution to this work was the development of a new, chloroform-resistant matrix for plasma desorption mass spectrometry with the use of the low molecular weight compound 3-(3-pyridyl) acrylic acid. With this matrix material a wide variety of different classes of compounds was investigated including peptides, glycopeptides and phospholipides. (author)

  8. Human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes

    Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is polyspecific as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies

  9. Recombinant Human Erythropoietin Protects Myocardial Cells from Apoptosis via the Janus-Activated Kinase 2/Signal Transducer and Activator of Transcription 5 Pathway in Rats with Epilepsy

    Bao-Xin Ma, MD

    2015-12-01

    Conclusions: These results indicate that myocardial cell apoptosis may contribute to myocardial injury in epilepsy. EPO protects myocardial cells from apoptosis via the JAK2/STAT5 pathway in rats with experimental epilepsy, whereas CEPO exerts antiapoptotic activity perhaps via a pathway independent of JAK2/STAT5 signaling.

  10. Containment air circulation for optimal hydrogen recombination

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  11. Selective desorption of carbon dioxide from sewage sludge for in-situ methane enrichment: Enrichment experiments in pilot scale

    The application of in-situ methane enrichment for upgrading the biogas from anaerobic digestion to vehicle fuel or natural gas quality, has been studied in pilot scale and by computer simulation of the desorption step. Pilot plant experiments have been performed using a 19 m3 and 15 m3 continuously stirred tank reactor operating with municipal sewage sludge at mesophilic conditions connected respectively to a 90 dm3 and 140 dm3 external bubble column for selective desorption of CO2. The results show that the CH4 yield is unchanged during the experiments, and accordingly there is no evidence that the oxygen in the air used in the desorption of CO2 has a negative impact on the CH4 producing activity. The sludge recirculation system must be designed to avoid leakage of air into the digester, in order to maintain a low N2 concentration in the biogas. At best, a biogas with a volume fraction of 87% CH4 and φN2 = 2% was obtained. The CH4 loss however amounted to 8%, which is unsatisfactory. The experimental results are compared with previous data in the literature and explanations are deducted for the difference in the performance. Computer simulations reveal that the sludge flow rate recirculated through the desorption column should be as low as possible to minimize the loss of CH4. An increased air flow rate through the desorption column and an increased desorption column volume will promote the desorption of CO2 and improve the ratio of CO2 to CH4 desorption. -- Highlights: ► In-situ methane enrichment for biogas upgrading. ► The methane yield is unchanged. ► Methane concentration of 87% obtained. ► The sludge flow rate should be low. ► The aeration rate should be high.

  12. Forced desorption of semiflexible polymers, adsorbed and driven by molecular motors

    Chaudhuri, Abhishek

    2014-01-01

    We formulate and characterize a model to describe dynamics of semiflexible polymers in the presence of activity due to motor proteins attached irreversibly to a substrate, and a transverse pulling force acting on one end of the filament. The stochastic binding-unbinding of the motor proteins and their ability to move along the polymer, generates active forces. As the pulling force reaches a threshold value, the polymer eventually desorbs from the substrate. We present a mean field theory that predicts increase in desorption force with polymer bending rigidity, active velocity and processivity of the motor proteins. Performing molecular dynamics simulations of the polymer in presence of a Langevin heat bath, and stochastic motor activity we obtain desorption phase diagrams that show good agreement with theory. With increase in pulling force, the polymer undergoes a first order phase transition from mostly adsorbed to fully desorbed state via a regime of coexistence where the steady state dynamics of the polyme...

  13. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

  14. Characterization of foot- and mouth disease virus antigen by surface-enhanced laser desorption ionization-time of flight-mass spectrometry in aqueous and oil-emulsion formulations

    Harmsen, M.M.; Jansen, J.; Westra, D.F.; Coco-Martin, J.M.

    2010-01-01

    We have used a novel method, surface-enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS), to characterize foot-and-mouth disease virus (FMDV) vaccine antigens. Using specific capture with FMDV binding recombinant antibody fragments and tryptic digestion of FMDV antig

  15. Integrated field emission array for ion desorption

    Resnick, Paul J; Hertz, Kristin L.; Holland, Christopher; Chichester, David

    2016-08-23

    An integrated field emission array for ion desorption includes an electrically conductive substrate; a dielectric layer lying over the electrically conductive substrate comprising a plurality of laterally separated cavities extending through the dielectric layer; a like plurality of conically-shaped emitter tips on posts, each emitter tip/post disposed concentrically within a laterally separated cavity and electrically contacting the substrate; and a gate electrode structure lying over the dielectric layer, including a like plurality of circular gate apertures, each gate aperture disposed concentrically above an emitter tip/post to provide a like plurality of annular gate electrodes and wherein the lower edge of each annular gate electrode proximate the like emitter tip/post is rounded. Also disclosed herein are methods for fabricating an integrated field emission array.

  16. Salt tolerance of desorption electrospray ionization (DESI).

    Jackson, Ayanna U; Talaty, Nari; Cooks, R Graham; Van Berkel, Gary J

    2007-12-01

    The salt tolerance of desorption electrospray ionization (DESI) was systematically investigated by examining three different drug mixtures in the presence of 0, 0.2, 2, 5, 10, and 20% NaCl:KCl (1:1) from different surfaces. At physiological salt concentrations, the individual drugs in each mixture were observed in each experiment. Even at salt concentrations significantly above physiological levels, particular surfaces were effective in providing spectra that allowed the ready identification of the compounds of interest in low nanogram amounts. Salt adducts, which are observed even in the absence of added salt, could be eliminated by adding 0.1% 7 M ammonium acetate to the standard methanol:water (1:1) spray solvent. Comparison of the salt tolerance of DESI with that of electrospray ionization (ESI) demonstrated better signal/noise characteristics for DESI. The already high salt tolerance of DESI can be optimized further by appropriate choices of surface and spray solution. PMID:17977744

  17. Coincidence experiments in desorption mass spectrometry

    Diehnelt, C. W.; English, R. D.; Van Stipdonk, M. J.; Schweikert, E. A.

    2002-06-01

    The detection of coincidental signals can enhance the amount of information available in desorption time-of-flight mass spectrometry (TOF-MS) by identifying physical, chemical and/or spatial correlations between secondary ions. Detection of coincidental emissions requires that the target surface be bombarded with individual primary ions (keV or MeV), each resolved in time and space. This paper will discuss the application of coincidence counting to TOF-MS to: extract the secondary ion mass spectrum and secondary ion yields from an organic target produced by a single primary ion type when multiple primary ions simultaneously impact the sample; examine the metastable dissociation pathways and decay fractions of organic secondary ions using an ion-neutral correlation method; and study the chemical microhomogeneity (on the sub-μm scale) of a surface composed of two chemically distinct species.

  18. Integrated field emission array for ion desorption

    Resnick, Paul J; Hertz, Kristin L; Holland, Christopher; Chichester, David; Schwoebel, Paul

    2013-09-17

    An integrated field emission array for ion desorption includes an electrically conductive substrate; a dielectric layer lying over the electrically conductive substrate comprising a plurality of laterally separated cavities extending through the dielectric layer; a like plurality of conically-shaped emitter tips on posts, each emitter tip/post disposed concentrically within a laterally separated cavity and electrically contacting the substrate; and a gate electrode structure lying over the dielectric layer, including a like plurality of circular gate apertures, each gate aperture disposed concentrically above an emitter tip/post to provide a like plurality of annular gate electrodes and wherein the lower edge of each annular gate electrode proximate the like emitter tip/post is rounded. Also disclosed herein are methods for fabricating an integrated field emission array.

  19. Uso de fator VII recombinante ativado para tratamento e profilaxia de grandes sangramentos Use of recombinant activated factor VII for treatment and prophylaxis of major bleeding

    Flávio Augusto Henriques Vince

    2009-09-01

    pacientes. Dessa forma, o recente aumento do uso de rFVIIa em situações ainda não aprovadas levou ao crescente questionamento sobre eficácia e segurança desta específica medicação em tais situações.INTRODUCTION: Recombinant activated factor VII (rFVIIa is a protein produced by genetic engineering, the structure is very similar to the structure of intrinsic activated factor VII (FVII. Its action is based on knowledge of the coagulation mechanism in vivo by acting in direct activation of factor X independent resulting in formation of thrombin at the injury site and thereby contributing to the formation of stable fibrin clots without the action of factor VIII and factor IX. METHODS: Was conducted extensive review of the literature in order to determine the new findings related to the use of recombinant activated factor VII in patients with severe bleeding. RESULTS: It was found that the use of rFVIIa started in the 80's for prophylaxis and treatment of bleeding in patients with a history of hemophilia A or B with inhibitors to factor VIII and IX, factor VII deficiency and Glanzmann's thrombasthenia refractory to replacement platelet. In 1999 its use was expanded to other clinical situations and thus began to be published several studies showing the efficacy of rFVIIa as a pro-hemostatic agent in patients with bleeding disorders or other previously healthy patients with a history of acute bleeding of major consequence. Trauma is the leading cause of mortality worldwide and uncontrolled bleeding the main challenge in caring for these patients. It is common for the association of trauma with coagulopathy, requiring in some cases specific therapy to treat it. At this point in adjuvant therapy with rFVIIa should be considered. Other common causes of bleeding are the heart, gynecologic/obstetric surgeries and diseases involving the liver. The coagulopathy in these cases is deficiency of factors dependent on vitamin K, and the FVII factor with smaller half life. CONCLUSION

  20. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-04-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s). PMID:26661793

  1. Recombinant erythropoietin for the anaemia of patients with advanced Gastrointestinal Stromal Tumours (GIST receiving imatinib: an active agent only in non progressive patients

    Duffaud Florence

    2012-09-01

    Full Text Available Abstract Recombinant erythropoietin for the anaemia of patients with advanced Gastrointestinal Stromal Tumours (GIST receiving imatinib : an active agent only in non progressive patients. Background Imatinib is a standard treatment for advanced/metastatic GIST and in adjuvant setting. Anaemia is frequently observed in patients with advanced GIST, and is one of the most frequent side effects of imatinib with grade 3–4 anaemia in 10% of patients. Whether EPO treatment is useful in the management of GIST patients receiving imatinib treatment is unknown. Methods A retrospective study of EPO treatment in GIST patients receiving imatinib was undertaken in 4 centres. Thirty four patients received EPO treatment among the 319 GIST patients treated with imatinib in clinical trials or with compassionate use between 2001 and 2003. The efficacy of EPO on the anaemia of patients with GIST treated with imatinib was analyzed. Results There were 18 males and 16 females with a median age of 59 years. Median WHO-PS was 1. Primary tumour sites were mainly gastric (32% and small bowel (29%. Sites of metastases were mainly liver (82% and peritoneum (79%. The median delay between the initiation of imatinib treatment and EPO was 58 days (range 0–553. Median haemoglobin (Hb level prior to EPO was 9 g/dL (range 6,9-11,8 and 11,7 g/dL (range 6,8-14,4 after 2 months. An increase of more than 2 g/dL was observed in 18 (53% of patients. None of the 7 patients who progressed (PD under imatinib treatment (400 mg/day experienced HB response, as compared to 66% (18/27 of the remaining patients (PR + SD (p = 0,002. Primary tumour site, liver metastases, peritoneal metastases, age, gender did not correlate with HB response to EPO. Response to EPO was observed in 2/11 patients receiving high-dose imatinib (800 mg/day vs 16/23 of others. Using logistic regression, only PD before EPO treatment was retained as a predictive factor for EPO response. Conclusion EPO enables to

  2. Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

    Brockelbank Jane A

    2007-01-01

    Full Text Available Abstract Background Fluorescence activated cell sorting (FACS is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. Results Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2 or the myelin oligodendrocyte glycoprotein (MOG fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin and the

  3. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

    Min-Ji Kim

    Full Text Available High-level expression of recombinant human growth hormone (hGH in Escherichia coli (E. coli leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC, size-exclusion chromatography (SEC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF mass spectrometry, and circular dichroism (CD. These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

  4. Damage, trapping and desorption at the implantation of helium and deuterium in graphite, diamond and silicon carbide

    The production, thermal stability and structure of ion induced defects have been studied by Rutherford backscattering in channeling geometry for the implantation of helium and deuterium in graphite, diamond and silicon carbide with energies of 8 and 20 keV. At the implantation of deuterium and helium ions more defects were measured in graphite than in diamond or silicon carbide at equal experimental conditions. This is due to increased backscattering in graphite, which is caused by the splitting and tilting of crystallites and a local reordering of lattice atoms around defects. At 300 K, Helium produces more defects in all three materials than deuterium with equal depth distribution of defects. The ratio of the defects produced by helium and deuterium agrees very well with the corresponding ratio of the energy deposited in nuclear collisions. In graphite, only small concentrations of deuterium induced defects anneal below 800 K, while in diamond small concentrations of deuterium as well as of helium induced defects anneal mostly below 800 K. This annealing behavior is considered to be due to recombination of point defects. The buildup of helium and deuterium in graphite is different. The trapping of deuterium proceeds until saturation is reached, while in the case of helium trapping is interrupted by flaking. In diamond, deuterium as well as helium are trapped almost completely until at higher fluences reemission starts and saturation is reached. Two desorption mechanisms were identified for the thermal desorption of helium from base-oriented graphite. Helium implanted at low fluences desorbs diffusing to the surface, while for the implantation of high fluences the release of helium due to blistering dominates. The desorption of deuterium from graphite and diamond shows differences. While in graphite the desorption starts already at 800 K, in diamond up to 1140 K only little desorption can be observed. These differences can be explained by the different transport

  5. Feasibility of desorption atmospheric pressure photoionization and desorption electrospray ionization mass spectrometry to monitor urinary steroid metabolites during pregnancy

    Vaikkinen, A.; Rejšek, Jan; Vrkoslav, Vladimír; Kauppila, T. J.; Cvačka, Josef; Kostiainen, R.

    2015-01-01

    Roč. 880, Jun 23 (2015), s. 84-92. ISSN 0003-2670 Grant ostatní: GA AV ČR(CZ) M200551204 Institutional support: RVO:61388963 Keywords : desorption electrospray ionization * desorption atmospheric pressure * photoionization * mass spectrometry * pregnancy Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.513, year: 2014

  6. Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

    Aizen, Joseph; Chandler, Jennifer C; Fitzgibbon, Quinn P; Sagi, Amir; Battaglene, Stephen C; Elizur, Abigail; Ventura, Tomer

    2016-04-01

    In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans. PMID:26883686

  7. Expression of Recombinant Antibodies

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  8. Adlayer structure dependent ultrafast desorption dynamics in carbon monoxide adsorbed on Pd (111)

    Hong, Sung-Young; Xu, Pan; Camillone, Nina R.; White, Michael G.; Camillone, Nicholas

    2016-07-01

    We report our ultrafast photoinduced desorption investigation of the coverage dependence of substrate-adsorbate energy transfer in carbon monoxide adlayers on the (111) surface of palladium. As the CO coverage is increased, the adsorption site population shifts from all threefold hollows (up to 0.33 ML), to bridge and near bridge (>0.5 to 0.6 ML) and finally to mixed threefold hollow plus top site (at saturation at 0.75 ML). We show that between 0.24 and 0.75 ML this progression of binding site motifs is accompanied by two remarkable features in the ultrafast photoinduced desorption of the adsorbates: (i) the desorption probability increases roughly two orders magnitude, and (ii) the adsorbate-substrate energy transfer rate observed in two-pulse correlation experiments varies nonmonotonically, having a minimum at intermediate coverages. Simulations using a phenomenological model to describe the adsorbate-substrate energy transfer in terms of frictional coupling indicate that these features are consistent with an adsorption-site dependent electron-mediated energy coupling strength, ηel, that decreases with binding site in the order: three-fold hollow > bridge and near bridge > top site. This weakening of ηel largely counterbalances the decrease in the desorption activation energy that accompanies this progression of adsorption site motifs, moderating what would otherwise be a rise of several orders of magnitude in the desorption probability. Within this framework, the observed energy transfer rate enhancement at saturation coverage is due to interadsorbate energy transfer from the copopulation of molecules bound in three-fold hollows to their top-site neighbors.

  9. Immune Response to Recombinant Capsid Proteins of Adenovirus in Humans: Antifiber and Anti-Penton Base Antibodies Have a Synergistic Effect on Neutralizing Activity

    Gahéry-Ségard, Hanne; Farace, Françoise; Godfrin, Dominique; Gaston, Jesintha; Lengagne, Renée; Tursz, Thomas; Boulanger, Pierre; Guillet, Jean-Gérard

    1998-01-01

    Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the vi...

  10. Workshop on Radio Recombination Lines

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  11. Monitoring of hydrogen absorption/desorption into pressed cobalt powder using resistometry

    The kinetics of hydrogen absorption/desorption into pressed cobalt powder samples was monitored using resistometry. The samples were prepared by pressing Co powder at 100 MPa and t = 25 oC. The equivalent diameter of the species varied from 5 to 200 μm. The study showed that hydrogen absorption leads to an increase in the electrical resistivity of the pressed samples. During diffusion into the crystal lattice, the absorbed hydrogen atoms simultaneously trap an electron from the conduction band of the metal and form H- ions, consequently inducing an increase in the electrical resistivity of the pressed sample. On the basis of the change in resistivity, a mechanism of hydrogen absorption has been proposed. During the initial period of absorption, the hydrogen absorption reaction is limited by the dissociative adsorption of hydrogen over a short period of time, following which the diffusion of H- ions into the bulk of the pressed Co powder is identified as the rate-determining step. The hydrogen desorption rate in an argon atmosphere is limited by H2,ad formation; recombination of adsorbed hydrogen atoms, Had, or by the diffusion of H- ions from the crystal lattice over a prolonged period of time.

  12. Adsorption and desorption performance of benzene over hierarchically structured carbon-silica aerogel composites.

    Dou, Baojuan; Li, Jinjun; Wang, Yufei; Wang, Hailin; Ma, Chunyan; Hao, Zhengping

    2011-11-30

    Hierarchically structured carbon-silica aerogel (CSA) composites were synthesized from cheap water glass precursors and granulated activated carbon via a post-synthesis surface modification with trimethylchlorosilane (TMCS) and a low-cost ambient pressure drying procedure. The resultant CSA composites possess micro/mesoporous structure and hydrophobic surface. The adsorption and desorption performance of benzene on carbon-silica aerogel composite (CSA-2) under static and dynamic conditions were investigated, comparing with pure silica aerogel (CSA-0) and microporous activated carbon (AC). It was found that CSA-2 has high affinity towards aromatic molecules and fast adsorption kinetics. Excellent performance of dynamic adsorption and desorption observed on CSA-2 is related to its higher adsorption capacity than CSA-0 and less mass transfer resistance than AC, arising from the well-developed microporosity and open foam mesostructure in the CSA composites. PMID:21962860

  13. Thermal desorption of Au from W(001) surface

    Blaszczyszyn, R; Godowski, P J

    2002-01-01

    Adsorption of Au on W(001) at 450 K up to multilayer structures was investigated. Temperature programmed desorption technique was used in determination of coverage dependent desorption energy (region up to one monolayer). Results were discussed in terms of competitive interactions of Au-Au and Au-W atoms. Simple procedure for prediction of faceting behavior on the interface, basing on the desorption data, was postulated. It was deduced that the Au/W(001) interface should not show faceting tendency after thermal treatment. (author)

  14. Thermal Desorption of Au from W(001) Surface

    Błaszczyszyn, R.; Chrzanowski, J.; Godowski, P. J.

    2000-12-01

    Adsorption of Au on W(001) at 450 K up to multilayer structures was investigated. Temperature programmed desorption technique was used in determination of coverage dependent desorption energy (region up to one monolayer). Results were discussed in terms of competitive interactions of Au--Au and Au--W atoms. Simple procedure for prediction of faceting behavior on the interface, basing on the desorption data, was postulated. It was deduced that the Au/W(001) interface should not show faceting tendency after thermal treatment.

  15. Desorption electrospray ionization using an Orbitrap mass spectrometer: Exact mass measurements on drugs and peptides.

    Hu, Qizhi; Talaty, Nari; Noll, Robert J; Cooks, R Graham

    2006-01-01

    Desorption electrospray ionization (DESI) is implemented on an Orbitrap mass spectrometer. The ion source is described and applications which utilize the high-resolution capabilities of the Orbitrap are emphasized, including the characterization of peptides and active ingredients in pharmaceutical tablets. Measurements are made in less than 1 s at a resolution of 60,000. The implications of the data for the mechanisms of DESI are discussed. PMID:17051609

  16. Silica-Gelatin Composite Materials for Prolonged Desorption of Bioactive Compounds

    Oksana STAVINSKAYA; Iryna LAGUTA; Inna OREL

    2014-01-01

    The set of gelatin and silica-gelatin films with incorporated bioactive compounds of different chemical nature was prepared. The swelling behavior of the films and desorption of active ingredients into aqueous media were studied. For all the silica-gelatin films we observed considerable improvement of the materials water stability and retardation of the release of bioactive compounds as compared to gelatin samples. The results obtained make it possible to consider silica-gelatin composite mat...

  17. Salt Tolerance of Desorption Electrospray Ionization (DESI)

    Jackson, Ayanna U. [Purdue University; Talaty, Nari [Purdue University; Cooks, R G [Purdue University; Van Berkel, Gary J [ORNL

    2007-01-01

    Suppression of ion intensity in the presence of high salt matrices is common in most mass spectrometry ionization techniques. Desorption electrospray ionization (DESI) is an ionization method that exhibits salt tolerance, and this is investigated. DESI analysis was performed on three different drug mixtures in the presence of 0, 0.2, 2, 5, 10, and 20% NaCl:KCl weight by volume from seven different surfaces. At physiological concentrations individual drugs in each mixture were observed with each surface. Collision-induced dissociation (CID) was used to provide additional confirmation for select compounds. Multiple stage experiments, to MS5, were performed for select compounds. Even in the absence of added salt, the benzodiazepine containing mixture yielded sodium and potassium adducts of carbamazepine which masked the ions of interest. These adducts were eliminated by adding 0.1% 7M ammonium acetate to the standard methanol:water (1:1) spray solvent. Comparison of the salt tolerance of DESI with that of electrospray ionization (ESI) demonstrated much better signal/noise characteristics for DESI in this study. The salt tolerance of DESI was also studied by performing limit of detection and dynamic range experiments. Even at a salt concentration significantly above physiological concentrations, select surfaces were effective in providing spectra that allowed the ready identification of the compounds of interest. The already high salt tolerance of DESI can be optimized further by appropriate choices of surface and spray solution.

  18. Regulation of Meiotic Recombination

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  19. Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis

    Sinha, Sushmita; Miller, Lisa; Subramanian, Sandhya; McCarty, Owen; Proctor, Thomas; Meza-Romero, Roberto; Burrows, Gregory G.; Vandenbark, Arthur A.; Offner, Halina

    2010-01-01

    Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in antigen specific manner. We evaluated effects of RTL401 (I-As α1β1 + PLP-139-151) on splenocytes from mice with EAE to study RTL- T cell-tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-α1β1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RT...

  20. Adsorption and desorption studies on hazardous dye Naphthol Yellow S

    In the present study, the batch technique was adopted under a variety of conditions, viz., amount of adsorbent, contact time, concentration, temperature and pH. By using UV spectrophotometer, concentration of dye was measured before and after adsorption. Dye removal data were fitted into the Langmuir and Freundlich adsorption isotherm equations. The values of their corresponding constants were determined. Thermodynamic parameters like free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) of the systems were calculated by using Langmuir constant. The estimated values for (ΔG) were -8.027 x 103 and -28.46 x 103 kJ mol-1 over activated carbon and activated de-oiled mustard at 303 K (30 oC), indicate toward a spontaneous process. The adsorption process followed pseudo-first-order model. The values of % removal and kad for dye systems were calculated at different temperatures ranging (303-323 K). Desorption studies indicate that elution by dilute NaOH through the fixed bed of the adsorbents columns could be regenerated and a quantitative recovery of Naphthol Yellow S can be achieved.

  1. Single-stranded heteroduplex intermediates in λ Red homologous recombination

    Zhang Youming

    2010-07-01

    Full Text Available Abstract Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

  2. Promoting and avoiding recombination: contrasting activities of the Escherichia coli RuvABC Holliday junction resolvase and RecG DNA translocase.

    Zhang, Jing; Mahdi, Akeel A; Briggs, Geoffrey S; Lloyd, Robert G

    2010-05-01

    RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC. PMID:20157002

  3. The Detection of Genotıxic Activity of The Deltamethrin and Permethrin by Somatic Mutation and Recombination Test With Drosophila Melanogaster

    F. Azize BUDAK

    2005-06-01

    Full Text Available In this study, different doses of deltamethrin from pyretyroide insecticides and permethrine from organophosphate insecticides have been analysied for their genotoxic effects by somatic mutation and recombination test (SMART in Drosophila melanogaster. Lethal doses of chemicals used were determined. Trans-heterozygous larvae for two genetic markers mwh and flr3 were evaluated for 25 ppm, 50 ppm and 75 ppm concentrations of the insecticides. A possitive correlation was observed among total mutations, the number of wings having mutations and percentage survival. Mutations observed were classified according to their size and type per wing and the dataevaluated by statistical analysis. Deltamethrin was found more toxic and mutagenic than permethrine.

  4. 重组活化人凝血因子Ⅶ在肝脏移植术中应用%Application of recombinant activated factor Ⅶ in liver transplantation

    李宏; 邓迺封

    2006-01-01

    重组活化人凝血因子Ⅶ(recombinant factor Ⅶa,rFⅦa),是一种新型止血药,最初用于治疗存在有因子Ⅷ(FⅧ)和因子IX(FIX)抗体(抑制物)的先天性血友病和继发性血友病患者的自发性或手术性出血。目前,已证实该药对控制多种临床出血情况是有益的。

  5. In vivo restoration of biologically active 3' ends of virus-associated RNAs by nonhomologous RNA recombination and replacement of a terminal motif.

    Carpenter, C. D.; Simon, A. E.

    1996-01-01

    Sequences at the 3' ends of plus-strand RNA viruses and their associated subviral RNAs are important cis elements for the synthesis of minus strands in vivo and in vitro. All RNAs associated with turnip crinkle virus (TCV), including the genomic RNA (4,054 bases) and satellite RNAs (sat-RNAs) such as sat-RNA D (194 bases), terminate with the motif CCUGCCC. While investigating the ability of in vivo-generated recombinants between sat-RNA D and TCV to be amplified in plants, we discovered that ...

  6. Reversed DNA Strand Cleavage Specificity in Initiation of Cre–LoxP Recombination Induced by the His289Ala Active-site Substitution

    Gelato, Kathy A.; Martin, Shelley S.; Baldwin, Enoch P.

    2010-01-01

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8 bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5′(S1 nucleotide) or 3′(S1′nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1′substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the “conformational switch” isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289

  7. Laser desorption mass spectrometry for biomolecule detection and its applications

    During the past few years, we developed and used laser desorption mass spectrometry for biomolecule detections. Matrix-assisted laser desorption/ionization (MALDI) was successfully used to detect DNA fragments with the size larger than 3000 base pairs. It was also successfully used to sequence DNA with both enzymatic and chemical degradation methods to produce DNA ladders. We also developed MALDI with fragmentation for direct DNA sequencing for short DNA probes. Since laser desorption mass spectrometry for DNA detection has the advantages of fast speed and no need of labeling, it has a great potential for molecular diagnosis for disease and person identification by DNA fingerprinting. We applied laser desorption mass spectrometry to succeed in the diagnosis of cystic fibrosis and several other nerve degenerative diseases such as Huntington's disease. We also succeeded in demonstrating DNA typing for forensic applications

  8. Molecular beam-thermal hydrogen desorption from palladium

    Among the most efficient techniques for hydrogen desorption monitoring, thermal desorption mass spectrometry is a very sensitive one, but in certain cases can give rise to uptake misleading results due to residual hydrogen partial pressure background variations. In this work one develops a novel thermal desorption variant based on the effusive molecular beam technique that represents a significant improvement in the accurate determination of hydrogen mass absorbed on a solid sample. The enhancement in the signal-to-noise ratio for trace hydrogen is on the order of 20%, and no previous calibration with a chemical standard is required. The kinetic information obtained from the hydrogen desorption mass spectra (at a constant heating rate of 1 deg. C/min) accounts for the consistency of the technique.

  9. Carbohydrate and steroid analysis by desorption electrospray ionization mass spectrometry.

    Kauppila, Tiina J; Talaty, Nari; Jackson, Ayanna U; Kotiaho, Tapio; Kostiainen, Risto; Cooks, R Graham

    2008-06-21

    Desorption electrospray ionization mass spectrometry (DESI-MS) is applied to the analysis of carbohydrates and steroids; the detection limits are significantly improved by the addition of low concentrations of salts to the spray solvent. PMID:18535704

  10. Influence of oxide layer on hydrogen desorption from zirconium hydride

    Hydrogen desorption from zirconium hydride with hydrogen content of 1.95 H/Zr in He and He-5%O2 atmospheres was studied by thermal desorption spectroscopy (TDS). The morphology, structure and valence states of the oxide layer formed on the surface of zirconium hydride were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). It was found that hydrogen desorption is retarded in the presence of oxygen. Heating in He leads to a large desorption range starting at 500 deg. C while heating in He-5%O2 atmosphere delays decomposition to relatively higher temperature of 525 deg. C. A protective oxide layer composed of monoclinic ZrO2 and small amount of tetragonal ZrO1.88, which acts as a very effective diffusion barrier. The O-H bond was observed in the oxide layer, which is beneficial to fix hydrogen atoms and prevent hydrogen diffusion

  11. Comparative sorption, desorption and leaching potential of aminocyclopyrachlor and picloram

    Aminocyclopyrachlor and picloram sorption, desorption and leaching potential were investigated in three soils from Minnesota and Hawaii. Aminocyclopyrachlor and picloram sorption fit the Freundlich equation and was independent of concentration for aminocyclopyrachlor (1/n = 1), but not for picloram ...

  12. 石斑鱼β-防御素的酵母表达及其产物抗菌活性分析%THE YEAST EXPRESSION OF GROUPER β-DEFENSIN AND ANTIBACTERIAL ACTIVITY OF THE RECOMBINANT PROTEIN

    金俊琰; 周莉; 桂建芳

    2011-01-01

    防御素是一类阳离子抗菌肽.研究从石斑鱼垂体SMART cDNA文库中扩增出129 bp石斑鱼β-防御素成熟肽序列,将其克隆到毕赤酵母表达载体pPCIZαA中,构建了石斑鱼β-防御素的真核表达载体,电击转化毕赤酵母GS115.Western Blot分析表明石斑鱼β-防御素在酵母菌中获得了表达.体外抗菌实验表明纯化的重组蛋白具有抑制大肠杆菌以及嗜水气单胞菌的作用,但是对革兰氏阳性菌,如金黄色葡萄球菌和藤黄微球菌的生长没有抑制作用.实验结果表明酵母表达的石斑鱼β-防御素能够特异地抑制革兰氏阴性菌的生长.%Defensin, a small cationic peptide, is an antimicrobial peptide, exhibit broad-spectrum antibacterial activity. They spread widely in plants, invertebrate and vertebrate animals, which can rapidly kill bacteria, fungi and viruses etc. Bacteria and viruses limited the production of grouper (Epinephelus coioides). In this study, we cloned grouper P-defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper P-defensin cDNA was amplified by polymerase chain reaction (PCR) from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper. The 129 bp DNA fragment encoding mature grouper P-defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPClZaA and transfected into Pichia pastoris GS115 expression host by electroporation. The genome of Pichia pastoris clone was extracted as template for screened the positive clone by using PCR. After grouper p-defensin was secreted by GS 115 by small-scale culture, the recombinant grouper P-defensin was induced by methanol for large scale. The supernatant was collected in 24h, 48h and 72h post induction for the recombinant protein detection. Subsequently, the grouper p-defensin peptide was detected in the supernatant of transfected yeast by Western Blot

  13. Recombination of chlorine atoms on plasma-conditioned stainless steel surfaces in the presence of adsorbed Cl2

    We investigated the interactions of atomic and molecular chlorine with plasma-conditioned stainless steel surfaces through both experiments and modelling. The recombination of Cl during adsorption and desorption of Cl2 was characterized using a rotating-substrate technique in which portions of the cylindrical substrate surface are periodically exposed to an inductively coupled chlorine plasma and then to an Auger electron spectrometer in separate, differentially pumped chambers. After several hours of exposure to the Cl2 plasma, the stainless steel substrate became coated with a Si-oxychloride-based layer (Fe : Si : O : Cl ∼ 1 : 13 : 13 : 3) due to chlorine adsorption and the erosion of the silica discharge tube. Desorption of Cl2 from this surface was monitored through measurements of pressure rises in the Auger chamber as a function of substrate rotation frequency. Significant adsorption and desorption of Cl2 was observed with the plasma off, similar to that observed previously on plasma-conditioned anodized aluminium surfaces, but with much faster desorption rates that are most likely attributable to the smoother and non-porous stainless steel surface morphology. When the plasma was turned on, a much larger pressure rise was observed due to Langmuir-Hinshelwood recombination of Cl atoms. Recombination coefficients, γCl, ranged from 0.004 to 0.03 and increased with Cl-to-Cl2 number density ratio. This behaviour was observed previously for anodized aluminium surfaces, and was explained by the blocking of Cl recombination sites by adsorbed Cl2. Application of this variable recombination coefficient to the modelling of high-density chlorine plasmas gives a much better agreement with measured Cl2 percent dissociations compared with predictions obtained with a recombination coefficient that is independent of plasma conditions.

  14. Nitrous oxides desorption from nitric acid (58–60 wt. %)

    Литвиненко, Олександр Олександрович; Печенко, Тамара Ивановна; Подустов, Михаил Алексеевич; Букатенко, Алексей Иванович

    2015-01-01

    The process of nitrous oxides desorption from nitric acid solutions (in domestic schemes) was considered. It is shown that in the process of desorption (or stripping), the nitrous oxides are not removed from solutions completely, so, the nitric acid does not satisfy the technical requirements in Ukraine.The research objective was to bring the quality of nitric acid to technological standards by removing (stripping) nitrous oxides from its solutions.To achieve the research objective, the balan...

  15. Study on hydrogen absorption/desorption properties of uranium alloys

    Ito, Hiroshi; Yamaguchi, Kenji; Yamawaki, Michio [Tokyo Univ., Tokai, Ibaraki (Japan). Nuclear Engineering Research Lab.

    1996-10-01

    Hydrogen absorption/desorption properties of two U-Mn intermetallic compounds, U{sub 6}Mn and UMn{sub 2}, were investigated. U{sub 6}Mn absorbed hydrogen and the hydrogen desorption pressure of U{sub 6}Mn obtained from this experiment was higher than that of U, which was considered to be the effect of alloying, whereas UMn{sub 2} was not observed to absorb hydrogen up to 50 atm at room temperature. (author)

  16. Recombination in ionized gases

    In this paper it is shown how capture-stabilized methodology (both macroscopic and microscopic) can provide a generic basis for a unified treatment of all of the above recombination mechanisms. A new semiclassical theory of dissociative recombination is also presented in an effort to gain further insight into the physics not included in the first-order treatment and difficult to extract from numerical quantal treatments based on configuration mixing and on multichannel quantum defect theory. A simple analytical expression more accurate than the standard first-order result is obtained for the cross section σ and rate coefficient α. (author)

  17. Water desorption of cassava starch granules: A study based on thermogravimetric analysis of aqueous suspensions and humid powders.

    Ayala Valencia, Germán; Djabourov, Madeleine; do Amaral Sobral, Paulo José

    2016-08-20

    This work reports on water desorption from cassava starch in relation with the structure and conditioning of granules in suspensions or after equilibration in desiccators. The experimental work is performed by thermogravimetric analysis with isothermal and non-isothermal protocols and interpreted to derive the activation energies and desorption frequencies according to the humidity range with no adjustable parameter. The analysis points out the different types of water interacting with the starch granules and relates the drying coefficients to their microscopic structure. The work helps clarifying contradictory and partial results from the literature. PMID:27178961

  18. Conceptual design for a full-scale VAC*TRAX vacuum thermal desorption unit. Final report, September 1992--December 1995

    Palmer, C.R.

    1996-04-01

    Rust Federal Services is pleased to present this topical report on the results of our Phase II conceptual design work of the PRDA VAC*TRAX{sup SM} mobile vacuum thermal desorption technology demonstration program. Through the present Phase II conceptual design activities, Rust has developed an equipment design and permitting strategy that retains the flexibility of a mobile treatment system with the long term value and ease of access of a central facility. The process is designed to remove volatile matter from solid matrices by thermal desorption. The system is also designed with superior emission controls, making it an ideal system for the treatment of radioactive wastes.

  19. Calcium lignosulfonate adsorption and desorption on Berea sandstone.

    Grigg, Reid B; Bai, Baojun

    2004-11-01

    This paper describes adsorption and desorption studies carried out with calcium lignosulfonate (CLS) on Berea sandstone. Circulation experiments were performed to determine CLS adsorption isotherms and the effects of CLS concentration, temperature, salinity, brine hardness, and injection rate on adsorption density. Flow-through experiments were performed to assess the reversibility of CLS adsorption and the influence of postflush rate, brine concentration, brine hardness, brine pH, and temperature on the desorption process. Results indicate that CLS adsorption isotherms on Berea sandstone follow the Freundlich isotherm law. The results presented in this paper on the effects of CLS adsorption and desorption on Berea sandstone show that: (1) increasing CLS concentration and salinity increases CLS adsorption density; (2) increasing temperature will decrease adsorption density; (3) increasing injection rate of CLS solution will slightly decrease CLS adsorption density; (4) postflush rate and salinity of brine have a large impact on the CLS desorption process; (5) the adsorption and desorption process are not completely reversible; and (5) temperature and pH of the postflush brine have little effect on desorption. PMID:15380409

  20. Sorption and desorption hysteresis of radiocesium on clay minerals

    The desorption of cesium on montmorillonite artificially loaded (3.36 mg/g) by humic acid and contaminated with cesium-137 solution 10-7 M CsCl[137Cs] was investigated. There was used repeated leaching technique (RLT) according to Kokotov and Popova. 6-8 leaching steps was accomplished to determine Kd and fraction of irreversibly sorbed (fixed) radiocesium Rfix values. A number of desorption solutions of various concentration and composition containing following competitive cations (K+, [Ag(tu)3]+, Na+, Ca2+, Fe3+, Fe2+, NH4+, Mg2+, Ba2+, Cu2+, Ni2+, Cr3+, Tl+) were examined. Rate of immobilised cesium is specific for each competitive cation, not depended on its concentration and the ratio of solid and liquid phases. Kd value drops with increasing concentration of desorption solution. Sorption and desorption of cesium on various clay minerals (illite, illite/smectite, hectorite, chlorite, vermiculite, kaolinite, montmorillonite) were investigated. Cation exchange capacity and Kd values were obtained. Sorption properties of cesium can be successfully described by the Sips isotherm. Kd and Rfix values were also obtained by desorption experiment using RLT method. Differences between the Kd values calculated for sorption and desorption steps were caused not only by various composition of solutions, but also by contribution of unknown Rfix at sorption. (authors)

  1. Sorption and desorption of 1,4-dichlorobenzene on peat

    2000-01-01

    Sorption and desorption of 1,4-dichlorobenzene (DCB) on peat (>92% organic matter)display large-scale hysteresis and nonlinearity. The magnitude of desorption hysteresis decreases in the order: untreated Pahokee peat (P)>acid treated peat (FP)>humin (TP). The desorption percentages are lower than 28% of the sorbed 1, 4-DCB after desorbing for 6 days. The sorption and desorption isotherms are well fitted to Freundlich equation, whose parameter 1/n ranges from 0.055 to 0.527. Moreover, the parameter 1/n of the desorption isotherm is significantly lower than that of the sorption isotherm, but the parameter IogK increases on contrary to 1/n. The desorption isotherms are very well fitted to Langmuir equation, whose Qm decreases in the order: TP>FP>P.The apparent partition coefficients (Kp) increase with increasing sorption time or decreasing aqueous equilibrium concentration of DCB. And Kp of P is significantly higher than that of FP or TP.

  2. Temperature dependence of CO desorption kinetics at a novel Pt-on-Au/C PEM fuel cell anode

    Pitois, A.; Pilenga, A.; Pfrang, A.; Tsotridis, G.; Abrams, Billie; Chorkendorff, Ib

    2010-01-01

    degrees C. The dependence in temperature of the desorption rate constants for the novel Pt-on-Au/C system is however much lower than that observed for the Pt/C system. This suggests that the nature of the substrate has a significant influence on the catalyst surface properties. It shows that, in surface-modified...... catalysts, the interactions between underlayer and overlayer materials are worthy of consideration, since they can significantly modify the intrinsic properties of the active sites. The kinetics of the CO desorption process have been discussed with regard to the CO tolerance issue at the PEM fuel cell anode.......A Pt-on-Au/C fuel cell anode catalyst has been obtained by electrochemical deposition of platinum on carbon-supported gold nanoparticles. Its composition, structure and nanoparticle size distribution have been characterised before and after the desorption experiments using microstructural...

  3. The role of electron-stimulated desorption in focused electron beam induced deposition

    van Dorp, Willem F.; Hansen, Thomas Willum; Wagner, Jakob Birkedal;

    2013-01-01

    We present the results of our study about the deposition rate of focused electron beam induced processing (FEBIP) as a function of the substrate temperature with the substrate being an electron-transparent amorphous carbon membrane. When W(CO)6 is used as a precursor it is observed that the growth...... experiments compared to literature values is consistent with earlier findings by other authors. The discrepancy is attributed to electron-stimulated desorption, which is known to occur during electron irradiation. The data suggest that, of the W(CO)6 molecules that are affected by the electron irradiation......, the majority desorbs from the surface rather than dissociates to contribute to the deposit. It is important to take this into account during FEBIP experiments, for instance when determining fundamental process parameters such as the activation energy for desorption....

  4. A Model of Hydrogen Desorption Kinetics Controlled both by Interface and Surface Reactions for Metal Hydrides

    Drozdov, I

    2014-01-01

    The desorption kinetics was modelled with the both interface- and surface reactions as rate-controlling steps. It has been shown analytically, that in the model of 'shrinking core' desorption, the finite hydride-decomposition-rate causes a modified slope of kinetics. The dependence of desorption time on the powder particle size has the same power of order as for the surface controlled desorption.

  5. Molecular Hydrogen Formation on Low Temperature Surfaces in Temperature Programmed Desorption Experiments

    Vidali, G; Li, L; Roser, J; Manico, G; Mehl, R; Lederhendler, A; Perets, H B; Brucato, J R; Biham, O

    2007-01-01

    The study of the formation of molecular hydrogen on low temperature surfaces is of interest both because it allows to explore elementary steps in the heterogeneous catalysis of a simple molecule and because of the applications in astrochemistry. Here we report results of experiments of molecular hydrogen formation on amorphous silicate surfaces using temperature-programmed desorption (TPD). In these experiments beams of H and D atoms are irradiated on the surface of an amorphous silicate sample. The desorption rate of HD molecules is monitored using a mass spectrometer during a subsequent TPD run. The results are analyzed using rate equations and the activation energies of the processes leading to molecular hydrogen formation are obtained from the TPD data. We show that a model based on a single isotope provides the correct results for the activation energies for diffusion and desorption of H atoms. These results can thus be used to evaluate the formation rate of H_2 on dust grains under the actual conditions...

  6. The Sorption/Desorption Behavior of Uranium in Transport Studies Using Yucca Mountain Alluvium

    Yucca Mountain, Nevada is the proposed site of a geologic repository for the disposal of spent nuclear fuel and high-level radioactive waste in the United States. In the event repository engineered barriers fail, the saturated alluvium located south of Yucca Mountain is expected to serve as a natural barrier to the migration of radionuclides to the accessible environment. The purpose of this study is to improve the characterization of uranium retardation in the saturated zone at Yucca Mountain to support refinement of an assessment model. The distribution of uranium desorption rates from alluvium obtained from Nye County bore holes EWDP-19IM1, EWDP-10SA, EWDP-22SA were studied to address inconsistencies between results from batch sorption and column transport experiments. The alluvium and groundwater were characterized to better understand the underlying mechanisms of the observed behavior. Desorption rate constants were obtained using an activity based mass balance equation and column desorption experiments were analyzed using a mathematical model utilizing multiple sorption sites with different first-order forward and reverse reaction rates. The uranium desorption rate constants decreased over time, suggesting that the alluvium has multiple types of active sorption sites with different affinities for uranium. While a significant fraction of the initially sorbed uranium desorbed from the alluvium quite rapidly, a roughly equivalent amount remained sorbed after several months of testing. The information obtained through this research suggests that uranium may experience greater effective retardation in the alluvium than simple batch sorption experiments would suggest. Electron Probe Microanalysis shows that uranium is associated with both clay minerals and iron oxides after sorption to alluvial material. These results provide further evidence that the alluvium contains multiple sorption sites for uranium

  7. The Sorption/Desorption Behavior of Uranium in Transport Studies Using Yucca Mountain Alluvium

    C. D. Scism

    2006-02-15

    Yucca Mountain, Nevada is the proposed site of a geologic repository for the disposal of spent nuclear fuel and high-level radioactive waste in the United States. In the event repository engineered barriers fail, the saturated alluvium located south of Yucca Mountain is expected to serve as a natural barrier to the migration of radionuclides to the accessible environment. The purpose of this study is to improve the characterization of uranium retardation in the saturated zone at Yucca Mountain to support refinement of an assessment model. The distribution of uranium desorption rates from alluvium obtained from Nye County bore holes EWDP-19IM1, EWDP-10SA, EWDP-22SA were studied to address inconsistencies between results from batch sorption and column transport experiments. The alluvium and groundwater were characterized to better understand the underlying mechanisms of the observed behavior. Desorption rate constants were obtained using an activity based mass balance equation and column desorption experiments were analyzed using a mathematical model utilizing multiple sorption sites with different first-order forward and reverse reaction rates. The uranium desorption rate constants decreased over time, suggesting that the alluvium has multiple types of active sorption sites with different affinities for uranium. While a significant fraction of the initially sorbed uranium desorbed from the alluvium quite rapidly, a roughly equivalent amount remained sorbed after several months of testing. The information obtained through this research suggests that uranium may experience greater effective retardation in the alluvium than simple batch sorption experiments would suggest. Electron Probe Microanalysis shows that uranium is associated with both clay minerals and iron oxides after sorption to alluvial material. These results provide further evidence that the alluvium contains multiple sorption sites for uranium.

  8. Recombinant DNA for Teachers.

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  9. Recombineering Pseudomonas syringae

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  10. Electrical Characteristics and Desorption Kinetics of Soil Boron

    2000-01-01

    The status and activities of boron in soils were studied by the approach of electro-ultrafiltration (EUF). The samples of soils, including brown-red soil and calcareous alluvial soil, were collected from Hubei Province of China. The soil samples were incubated in saturated water and then their nutrients were ultrafiltrated with EUF equipment. Filtration and extraction were conducted in accordance with routine process, but fractions in anode and cathode were all collected. Analyses of B, K+, Mg2+, Ca2+, C1- and pH in fractions supposed that boron existed not only in a simple form of borate but also in ion-pair with cations partly in acidic soil,and borate was the primary form existing in the calcareous soil. In studying desorption kinetics with EUF,the boron content of Fractions 2~6 was accumulated, and the accumulative quantities were fit to time factors in three kinetic equations: the zero-order, first-order, and arabolic diffusion equations. Fit degree of the parabolic diffusion equation was the best, followed by the zero-order quation, and the first-order equation was the worst.

  11. Desorption electrospray ionization of explosives on surfaces: sensitivity and selectivity enhancement by reactive desorption electrospray ionization.

    Cotte-Rodríguez, Ismael; Takáts, Zoltán; Talaty, Nari; Chen, Huanwen; Cooks, R Graham

    2005-11-01

    Desorption electrospray ionization (DESI), an ambient mass spectrometry technique, is used for trace detection of the explosives trinitrohexahydro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 2,4,6-trinitrotoluene (TNT), Pentaerythritol tetranitrate (PETN), and their plastic compositions (Composition C-4, Semtex-H, Detasheet) directly from a wide variety of surfaces (metal, plastic, paper, polymer) without sample preparation or pretreatment. Analysis of the explosives is performed under ambient conditions from virtually any surface in very short times (<5 s) including confirmatory tandem mass spectrometry (MS/MS) experiments, while retaining the sensitivity and specificity that mass spectrometry offers. Increased selectivity is obtained both by MS/MS and by performing additional experiments in which additives are included in the spray solvent. These reactive DESI experiments (reactions accompanying desorption) produce such ions as the chloride and trifluoroacetate adducts of RDX and HMX or the Meisenheimer complex of TNT. Desorption atmospheric pressure chemical ionization, a variant of DESI that uses gas-phase ions generated by atmospheric pressure corona discharges of toluene or other organic compounds, provides evidence for a heterogeneous-phase (gaseous ion/absorbed analyte) charge-transfer mechanism of DESI ionization in the case of explosives. Plastic explosives on surfaces were analyzed directly as fingerprints, without sample preparation, to test DESI as a possible method for in situ detection of explosives-contaminated surfaces. DESI also allowed detection of explosives in complex matrixes, including lubricants, household cleaners, vinegar, and diesel fuel. Absolute limits of detection for the neat explosives were subnanogram in all cases and subpicogram in the case of TNT. The DESI response was linear over 3 orders of magnitude for TNT. Quantification of RDX on paper gave a precision (RSD) of 2.3%. Pure water could be used

  12. Data on the evolutionary history of the V(D)J recombination-activating protein 1 - RAG1 coupled with sequence and variant analyses.

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Muppavarapu, Sekhar; Tandon, Ravi

    2016-09-01

    RAG1 protein is one of the key component of RAG complex regulating the V(D)J recombination. There are only few studies for RAG1 concerning evolutionary history, detailed sequence and mutational hotspots. Herein, we present out datasets used for the recent comprehensive study of RAG1 based on sequence, phylogenetic and genetic variant analyses (Kumar et al., 2015) [1]. Protein sequence alignment helped in characterizing the conserved domains and regions of RAG1. It also aided in unraveling ancestral RAG1 in the sea urchin. Human genetic variant analyses revealed 751 mutational hotspots, located both in the coding and the non-coding regions. For further analysis and discussion, see (Kumar et al., 2015) [1]. PMID:27284568

  13. Rabbits immunized with Epstein-Barr virus gH/gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350.

    Cui, Xinle; Cao, Zhouhong; Chen, Quanyi; Arjunaraja, Swadhinya; Snow, Andrew L; Snapper, Clifford M

    2016-07-25

    Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been strongly implicated in the etiology of multiple epithelial and lymphoid cancers, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma and post-transplant lymphoproliferative disorder. There is currently no licensed prophylactic vaccine for EBV. Most efforts to develop prophylactic vaccines have focused on EBV gp350, which binds to CD21/CD35 to gain entry into B cells, and is a major target of serum neutralizing antibody in EBV seropositive humans. However, a recombinant monomeric gp350 protein failed to prevent EBV infection in a phase II clinical trial. Thus, alternative or additional target antigens may be necessary for a successful prophylactic vaccine. EBV gH/gL and gB proteins coordinately mediate EBV fusion and entry into B cells and epithelial cells, strongly suggesting that vaccination with these proteins might elicit antibodies that will prevent EBV infection. We produced recombinant trimeric and monomeric EBV gH/gL heterodimeric proteins and a trimeric EBV gB protein, in addition to tetrameric and monomeric gp350(1-470) proteins, in Chinese hamster ovary cells. We demonstrated that vaccination of rabbits with trimeric and monomeric gH/gL, trimeric gB, and tetrameric gp350(1-470) induced serum EBV-neutralizing titers, using cultured human B cells, that were >100-fold, 20-fold, 18-fold, and 4-fold higher, respectively, than monomeric gp350(1-470). These data strongly suggest a role for testing EBV gH/gL and EBV gB in a future prophylactic vaccine to prevent EBV infection of B cells, as well as epithelial cells. PMID:27291087

  14. Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

    Johnson, T M; Pease, E A; Li, J K; Tien, M

    1992-08-01

    Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification. PMID:1632652

  15. Adsorption and desorption dynamics of citric acid anions in soil

    Oburger, E.

    2011-07-26

    The functional role of organic acid anions in soil has been intensively investigated, with special focus on (i) microbial respiration and soil carbon dynamics, (ii) nutrient solubilization or (iii) metal detoxification and reduction of plant metal uptake. Little is known about the interaction dynamics of organic acid anions with the soil matrix and the potential impact of adsorption and desorption processes on the functional significance of these effects. The aim of this study was to characterize experimentally the adsorption and desorption dynamics of organic acid anions in five agricultural soils differing in iron and aluminium oxide contents and using citrate as a model carboxylate. Results showed that both adsorption and desorption processes were fast in all soils, reaching a steady state within approximately 1 hour. However, for a given total soil citrate concentration (ct) the steady state was critically dependent on the starting conditions of the experiment, whether most of the citrate was initially present in solution (cl) or held on the solid phase (cs). Specifically, desorption-led processes resulted in significantly smaller steady-state solution concentrations than adsorption-led processes, indicating that hysteresis occurred. As it is not possible to distinguish between different adsorption and desorption pools in soil experimentally, a new dynamic hysteresis model that relies only on measured soil solution concentrations was developed. The model satisfactorily explained experimental data and was able to predict dynamic adsorption and desorption behaviour. To demonstrate its use, we applied the model to two relevant situations involving exudation and microbial degradation. The study highlighted the complex nature of citrate adsorption and desorption dynamics in soil. We conclude that existing models need to incorporate both temporal and hysteresis components to describe realistically the role and fate of organic acids in soil processes. © 2011 The

  16. Atomic Oxygen Desorption from an Amorphous Silicate Surface

    He, Jiao; Vidali, Gianfranco

    2014-06-01

    Oxygen is the third most abundant element in space. How oxygen-containing molecules form in space, and whether they form through gas-phase or grain-surface reactions, depends largely on the availability of atomic oxygen in gas-phase versus on surfaces of dust grains. The relative abundance of O in gas-phase versus on grain surfaces is determined by the residence time, or equivalently, desorption energy, of atomic oxygen on grain surfaces. Though important in astrochemical modeling, experimental investigations of atomic oxygen desorption from grain surfaces are lacking in the literature. In most astrochemical models, the O desorption energy value has been taken to be 800 K, which is a guessed value without experimental support. Based on this value, the predicted molecular oxygen abundance in space is at least 2 orders of magnitude higher than what space observations have found. This long running discrepancy of molecular oxygen abundance could be resolved if the O desorption energy is twice as the widely used value (Melnick, G., Tolls, V., et al. 2012, Astrophys. J., 752, 26). We performed TPD (thermal programmed desorption) experiments to study the ozone formation process via O+O2 on an amorphous silicate surface that emulates interstellar conditions. A rate equation model was used to characterize the surface kinetics of both atomic and molecular oxygen. The O desorption energy was extracted from rate equation simulations that best fit the TPD data. The value was found to be 1764±232 K, which agrees with what Melnick et al. proposed. We suggest that the newly found value for the O desorption energy should be used in astrochemical modeling. This work is supported by NSF, Astronomy & Astrophysics Division (Grants No. 0908108 and 1311958), and NASA (Grant No. NNX12AF38G). We thank Dr. J.Brucato of the Astrophysical Observatory of Arcetri for providing the samples used in these experiments.

  17. Influence of acid rain components on radiocesium-137 desorption from Cetraria islandica (L. Ach. lichen

    Miljanić Šćepan S.

    2012-01-01

    Full Text Available Desorption of 137Cs from Cetraria islandica (L. Ach. lichen was performed by five consecutive desorptions with five identical solution volumes. Solutions of H2SO4, HNO3 and their mixtures, with pH 4.61, 5.15 and 5.75 were used for desorption. The desorbed amount of 137Cs (average value, all solutions used from lichen, for a given pH value was 49.2% for pH 4.61; 47.0% for pH 5.15 and 47.6% for pH 5.75. The obtained values of the desorbed amount of 137Cs from lichen are in accordance with the data obtained in earlier work, when 46.2 % 137Cs was desorbed from lichen for pH 3.75, and 47.2% was desorbed for pH 2.87. A higher percentage of 59.8%, obtained for pH 2.00 indicates increased activity of H+ ions. The amount of desorbed 137Cs from lichen using solutions corresponding to acid rain cannot be lower than the stated values as they contain other substances besides the acid solutions used in this work.

  18. Sorption / desorption hysteresis of non-polar organic compounds in carbonaceous materials

    Wang, G.; Kleineidam, S.; Grathwohl, P.

    2003-04-01

    Natural and thermally-altered carbonaceous materials (coals, charcoal, cokes) are nowadays found in many soil and sediment samples due to anthropogenic impact (e.g. industrial activities, traffic) to the natural environment. The sorption -- desorption characteristics of hydrophobic organic compounds on that type of geosorbents play an important role in contaminant fate and transport and are yet not precisely known. Contaminant transport and bioavailibility models often assume complete sorption reversibility. In this study, a series of desorption steps using the ASE (accelerated solvent extractor) with water at different temperatures were carried out to determine the desorption enthalpy and to study hysteresis phenomena. The initial natural loading of PAHs onto the geosorbents was determined by solvent extraction (acetone and toluene). The background loading is in the order of 0.8 mg kg-1 -- 102 mg kg-1 for the natural coals and in the order of 0.16 mg kg-1 -- 10 mg kg-1 for the thermally altered coals. The results are compared to kinetic and equilibrium data from laboratory experiments on sorptive uptake of the phenanthrene onto the same carbonaceous samples.

  19. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  20. Human recombinant lysosomal enzymes produced in microorganisms.

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  1. Sorption-desorption dynamics of radiocaesium in organic matter soils

    A systematic study has been carried out on the radiocaesium sorption properties of 25 soils (forest, peat) covering organic matter (OM) contents in the range of 10-97%. Predictions are made for radiocaesium partitioning between micaceous Frayed Edge Sites (FES) and regular exchange sites (RES) on the basis of specific radiocaesium interception potentials of the soil and overall exchange capacity. It is shown that for soils with a very high OM content (>80%), significant fractions are present in a readily reversible form in the OM phase. In soils of low-medium OM content (<40%), only a very minor fraction is present in the OM exchange complex. Experimental findings, based on a desorption screening with a variety of desorption agents are in agreement with these predictions. On the basis of a study of sorption kinetics, some additional tools are available for identifying problem soils. In cases of very high OM content, radiocaesium adsorption is completed within hours demonstrating the involvement of the OM sites. In soils for which interception occurs in the FES, sorption continues to proceed for periods of 2-3 weeks. In conclusion, some examples are presented on radiocaesium desorption using ion exchangers as radiocaesium sinks in promoting desorption. For a peaty soil, near quantitative desorption is accomplished. For forest soils with OM contents in a range of 10-40%, fixation levels of 30-50% are demonstrated

  2. Cesium sorption and desorption behavior of clay minerals

    Cesium sorption and desorption of clay minerals (montmorillonite, beidellite, nontronite, weathered biotite, rectorite and illite) were investigated by consecutive sorption-desorption (CSD) experiments. In batch sorption experiment, two solutions with different Cs concentration 10-3 and 10-7 mol/L) were used. In batch desorption experiments, Cs sorbed samples in sorption experiments were treated 5 times with 1 mol/L ammonium acetate solution. In the case of CSD experiments using 10-3 mol/L Cs solution, the exchangeable cations (Na, Ca, and K) in the clay samples affected to the sorption ratio of Cs, and this effect depended on the type of clay mineral. The desorption ratios of untreated, Na-exchanged and Ca-exchanged weathered biotite ranged from 23 to 33%, while that of other samples was over 80%. In the case of CSD experiments using 10-7 mol/L Cs solution, the sorption ratio of montmorillonite was smaller than that of the other clay samples. In desorption experiments, more than 10-9 mol sorbed Cs remained in 1.0 g of the sample after 5 extraction times. These results indicate that all examined clay samples are able to strongly adsorb Cs with a capacity of more than 10-9 mol/g. (author)

  3. Sorption-desorption behavior of polybrominated diphenyl ethers in soils

    Polybrominated diphenyl ethers (PBDEs) are flame retardants that are commonly found in commercial and household products. These compounds are considered persistent organic pollutants. In this study, we used 4,4'-dibromodiphenyl ether (BDE-15) as a model compound to elucidate the sorption and desorption behavior of PBDEs in soils. The organic carbon-normalized sorption coefficient (KOC) of BDE-15 was more than three times higher for humin than for bulk soils. However, pronounced desorption hysteresis was obtained mainly for bulk soils. For humin, increasing concentration of sorbed BDE-15 resulted in decreased desorption. Our data illustrate that BDE-15 and probably other PBDEs exhibit high sorption affinity to soils. Moreover, sorption is irreversible and thus PBDEs can potentially accumulate in the topsoil layer. We also suggest that although humin is probably a major sorbent for PBDEs in soils, other humic materials are also responsible for their sequestration. - Highlights: → BDE-15 exhibited pronounced desorption hysteresis. → BDE-15 sowed higher sorption affinity to humin as compared to the bulk soils. → Sequestration of PBDEs depends on soil organic matter constitutes other than humin. - Pronounced desorption hysteresis was observed for BDE-15 in natural soils.

  4. Influence of surface coverage on the chemical desorption process

    Minissale, M.; Dulieu, F., E-mail: francois.dulieu@obspm.fr [LERMA, Université de Cergy Pontoise et Observatoire de Paris, UMR 8112 du CNRS. 5, mail Gay Lussac, 95031 Cergy Pontoise (France)

    2014-07-07

    In cold astrophysical environments, some molecules are observed in the gas phase whereas they should have been depleted, frozen on dust grains. In order to solve this problem, astrochemists have proposed that a fraction of molecules synthesized on the surface of dust grains could desorb just after their formation. Recently the chemical desorption process has been demonstrated experimentally, but the key parameters at play have not yet been fully understood. In this article, we propose a new procedure to analyze the ratio of di-oxygen and ozone synthesized after O atoms adsorption on oxidized graphite. We demonstrate that the chemical desorption efficiency of the two reaction paths (O+O and O+O{sub 2}) is different by one order of magnitude. We show the importance of the surface coverage: for the O+O reaction, the chemical desorption efficiency is close to 80% at zero coverage and tends to zero at one monolayer coverage. The coverage dependence of O+O chemical desorption is proved by varying the amount of pre-adsorbed N{sub 2} on the substrate from 0 to 1.5 ML. Finally, we discuss the relevance of the different physical parameters that could play a role in the chemical desorption process: binding energy, enthalpy of formation, and energy transfer from the new molecule to the surface or to other adsorbates.

  5. Sorption-desorption behavior of polybrominated diphenyl ethers in soils

    Olshansky, Yaniv; Polubesova, Tamara [Department of Soil and Water Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel); Vetter, Walter [Institute of Food Chemistry (170b), University of Hohenheim, Garbenstr. 28, D-70599 Stuttgart (Germany); Chefetz, Benny, E-mail: chefetz@agri.huji.ac.il [Department of Soil and Water Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel)

    2011-10-15

    Polybrominated diphenyl ethers (PBDEs) are flame retardants that are commonly found in commercial and household products. These compounds are considered persistent organic pollutants. In this study, we used 4,4'-dibromodiphenyl ether (BDE-15) as a model compound to elucidate the sorption and desorption behavior of PBDEs in soils. The organic carbon-normalized sorption coefficient (K{sub OC}) of BDE-15 was more than three times higher for humin than for bulk soils. However, pronounced desorption hysteresis was obtained mainly for bulk soils. For humin, increasing concentration of sorbed BDE-15 resulted in decreased desorption. Our data illustrate that BDE-15 and probably other PBDEs exhibit high sorption affinity to soils. Moreover, sorption is irreversible and thus PBDEs can potentially accumulate in the topsoil layer. We also suggest that although humin is probably a major sorbent for PBDEs in soils, other humic materials are also responsible for their sequestration. - Highlights: > BDE-15 exhibited pronounced desorption hysteresis. > BDE-15 sowed higher sorption affinity to humin as compared to the bulk soils. > Sequestration of PBDEs depends on soil organic matter constitutes other than humin. - Pronounced desorption hysteresis was observed for BDE-15 in natural soils.

  6. Synchrotron Radiation-Induced Desorption from a NEG-Coated Vacuum Chamber

    Chiggiato, P

    1999-01-01

    When the whole inner surface of a vacuum chamber is coated with a non-evaporable getter film, very low static and dynamic pressures are expected after activation. In an accelerator environment this could result in a longer beam lifetime, in a lower risk of pressure bumps, and in a lower level of bremsstrahlung radiation due to the beam-gas interactions. To substantiate these favourable characteristics a Ti-Zr-V coated stainless steel chamber has been tested on a dedicated beamline at the ESRF. It is shown that a large reduction of the synchrotron radiation-induced desorption occurs after activation.

  7. Synchrotron radiation-induced desorption from a NEG-coated vacuum chamber [of ESRF beamline

    Chiggiato, P

    2001-01-01

    When the whole inner surface of a vacuum chamber is coated with a non-evaporable getter film, very low static and dynamic pressures are expected after activation. In an accelerator environment this could result in a longer beam lifetime, in a lower risk of pressure bumps, and in a lower level of bremsstrahlung radiation due to the beam-gas interactions. To substantiate these favourable characteristics a Ti- Zr-V coated stainless-steel chamber has been tested on a dedicated beamline at the ESRF. It is shown that a large reduction of the synchrotron radiation-induced desorption occurs after activation. (10 refs).

  8. Recombinant influenza vaccines.

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  9. RECOMBINANT INFLUENZA VACCINES

    Sedova, E.; Shcherbinin, D.; Migunov, A.; Smirnov, Iu; Logunov, D.; Shmarov, M.; Tsybalova, L.; Naroditskiĭ, B.; O. Kiselev; Gintsburg, A.

    2012-01-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery pla...

  10. Nonradiative recombination in semiconductors

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  11. Recombinant human bone morphogenetic protein induces bone formation

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  12. The Effect of Recombinant Ghrelin Plasmid on Pepsin and Proton Pump Activity in Rats%重组生长素对大鼠胃蛋白酶和质子泵活性的影响

    杜改梅; 晏文梅; 胡志华; 蒋加进; 罗碧平

    2012-01-01

    10 weaned rats are randomly divided into treatment and control groups to explore the impact of recombinant ghrelin plasmid on pepsin and proton pump activity. After 5 days, rats in the treatment group are injected with recombinant human growth hormone in the leg muscle. The research is lasted for 29 days. Rats are killed after the test is complete. The stomach is separated and weighed. The gastric mucosa is taken for pepsin and proton activity. The results show that the pepsin and proton pump activity are significantly increased in the treatment group. Therefore, ghrelin can promote gastric exocrine function.%研究生长素对大鼠胃蛋白酶和质子泵活性的影响。取10只刚断乳大鼠,随机分为试验组和对照组。预饲5d后,给试验组大鼠腿部肌肉注射重组生长紊,试验29d。试验结束后处死大鼠,分离胃,称重,取胃粘膜组织测定质子泵活性和胃蛋白酶活性。结果发现试验组大鼠质子泵和胃蛋白酶活性显著高于对照组,提示Ghrelin可以促进对胃外分泌功能具有重要的调节作用。

  13. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    陆华中; 陈立; 王红卫; 伍志坚; 吴小兵; 王学峰; 王鸿利; 卢大儒; 邱信芳; 薛京伦

    2001-01-01

    A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

  14. Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy.

    Mealey, Robert H; Littke, Matt H; Leib, Steven R; Davis, William C; McGuire, Travis C

    2008-01-15

    Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000U/m2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2. PMID:17727961

  15. Thermal desorption analysis of beryllium tile pieces from JET

    Pieces of beryllium tile exposed to a D-D plasma in JET have been studied by thermal desorption spectroscopy. These tiles have a thick layer of redeposited Be-C-O with considerable hydrogen and deuterium present. The samples were heated at a constant rate of 2 C/min. from 100 C to 900 C. Desorption peaks occurred in the range of 140-480 C. There was no significant desorption at temperatures above 600 C. The amount of deuterium detected varied from a low of 8 x 1021/m2 to a high of 2.1 x 1023/m2. In one case, the amount of deuterium in a tile piece was seven times greater than the amount in a neighboring tile piece. Some of the tile pieces in the plasma-exposed region showed surface melting. Despite this, the deuterium yield from one of these pices is >1023/m2. (orig.)

  16. Desorption Mass Spectrometry for Nonvolatile Compounds Using an Ultrasonic Cutter

    Habib, Ahsan; Ninomiya, Satoshi; Chen, Lee Chuin; Usmanov, Dilshadbek T.; Hiraoka, Kenzo

    2014-07-01

    In this work, desorption of nonvolatile analytes induced by friction was studied. The nonvolatile compounds deposited on the perfluoroalkoxy substrate were gently touched by an ultrasonic cutter oscillating with a frequency of 40 kHz. The desorbed molecules were ionized by a dielectric barrier discharge (DBD) ion source. Efficient desorption of samples such as drugs, pharmaceuticals, amino acids, and explosives was observed. The limits of detection for these compounds were about 1 ng. Many compounds were detected in their protonated forms without undergoing significant fragmentation. When the DBD was off, no ions for the neutral samples could be detected, meaning that only desorption along with little ionization took place by the present technique.

  17. Sorption and desorption of tritiated water on paints

    In this paper the sorption and desorption of tritiated water on paints, such as acryl, silicone, epoxy and fluorine types, has been studied. In dynamic adsorption and desorption experiments, tritium concentration in the chamber, in which painted metal was installed, was measured during HTO vapor charging or purging by N2 + H2O. Slow desorption rate for acryl type was observed. Residual tritium not released by sweeping with N2 + H2O was recovered by heating up to 800 C. Residual tritium fraction for epoxy type was found to be highest among tested. Paint membrane was used in permeation experiments of HTO vapor and liquid. Diffusivity, solubility and permeability were evaluated. Diffusivity for fluorine was largest and that for epoxy smallest

  18. Hydrogen retention in tungsten materials studied by Laser Induced Desorption

    Zlobinski, M.; Philipps, V.; Schweer, B.; Huber, A.; Reinhart, M.; Möller, S.; Sergienko, G.; Samm, U.; 't Hoen, M. H. J.; Manhard, A.; Schmid, K.; Textor Team

    2013-07-01

    Development of methods to characterise the first wall in ITER and future fusion devices without removal of wall tiles is important to support safety assessments for tritium retention and dust production and to understand plasma wall processes in general. Laser based techniques are presently under investigation to provide these requirements, among which Laser Induced Desorption Spectroscopy (LIDS) is proposed to measure the deuterium and tritium load of the plasma facing surfaces by thermal desorption and spectroscopic detection of the desorbed fuel in the edge of the fusion plasma. The method relies on its capability to desorb the hydrogen isotopes in a laser heated spot. The application of LID on bulk tungsten targets exposed to a wide range of deuterium fluxes, fluences and impact energies under different surface temperatures is investigated in this paper. The results are compared with Thermal Desorption Spectrometry (TDS), Nuclear Reaction Analysis (NRA) and a diffusion model.

  19. A study of the process of desorption of hexavalent chromium

    W.B. Amorim

    2003-09-01

    Full Text Available In this work the process of desorption of hexavalent chromium, a toxic metal ion, from the marine algae Sargassum sp, following biosorption experiments 2³ factorial design was studied. A technique was applied to three eluents: HCl, H2SO4 and EDTA. Three factors of importance were evaluated: concentration of eluent, the ratio between mass of biosorbent and volume of eluent (S/L and process time. A statistical analysis of the experimental results showed that the three variables evaluated are significant for all three eluents. The models for chromium desorption were validated, as the results agreed well with the observed values. Through use of the response surface methodology, a factorial design based optimization technique; it was possible to identify the most suitable eluent and the interval of values for the process variables that resulted in the most significant desorption of chromium, which is relevant information for work aiming at process optimization.

  20. Desorption Dynamics, Internal Energies and Imaging of Organic Molecules from Surfaces with Laser Desorption and Vacuum Ultraviolet (VUV) Photoionization

    Kostko, Oleg; Takahashi, Lynelle K.; Ahmed, Musahid

    2011-04-05

    There is enormous interest in visualizing the chemical composition of organic material that comprises our world. A convenient method to obtain molecular information with high spatial resolution is imaging mass spectrometry. However, the internal energy deposited within molecules upon transfer to the gas phase from a surface can lead to increased fragmentation and to complications in analysis of mass spectra. Here it is shown that in laser desorption with postionization by tunable vacuum ultraviolet (VUV) radiation, the internal energy gained during laser desorption leads to minimal fragmentation of DNA bases. The internal temperature of laser-desorbed triacontane molecules approaches 670 K, whereas the internal temperature of thymine is 800 K. A synchrotron-based VUV postionization technique for determining translational temperatures reveals that biomolecules have translational temperatures in the range of 216-346 K. The observed low translational temperatures, as well as their decrease with increased desorption laser power is explained by collisional cooling. An example of imaging mass spectrometry on an organic polymer, using laser desorption VUV postionization shows 5 mu m feature details while using a 30 mu m laser spot size and 7 ns duration. Applications of laser desorption postionization to the analysis of cellulose, lignin and humic acids are briefly discussed.