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Sample records for ma104 cells trypsin-activated

  1. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    International Nuclear Information System (INIS)

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B.; Rubin, D.H.

    1988-01-01

    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated 51 Cr, [ 14 C]choline, and [ 3 H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration

  2. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. (Stanford Univ. School of Medicine, CA (USA) Palo Alto Veterans Administration Medical Center, CA (USA)); Rubin, D.H. (Univ. of Pennsylvania, Philadelphia (USA))

    1988-04-01

    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  3. Effects of bisphenol S on the structures and activities of trypsin and pepsin.

    Science.gov (United States)

    Wang, Yan-Qing; Zhang, Hong-Mei

    2014-11-19

    The effects of bisphenol S on the structures and activities of trypsin and pepsin were investigated by various methods like UV-visible absorbance, fluorescence, circular dichroism, and molecular docking. The secondary and tertiary structures of trypsin and pepsin were altered by bisphenol S binding, which resulted in the loosening of the skeletons of trypsin and pepsin. In addition, bisphenol S induced microenvironmental changes around tyrosine and tryptophan residues of trypsin and pepsin. The activity experimental results showed that the activity of pepsin decreases obviously with the increasing concentration of BPS, while the activity of trypsin does not change remarkably. The binding and thermodynamic parameters obtained by molecular docking and fluorescence spectroscopy showed that the bindings of bisphenol S to trypsin and pepsin were spontaneous processes and hydrogen bonding and hydrophobic interactions played a vital role in stabilizing the bisphenol S-trypsin and bisphenol S-pepsin complexes. The binding constants (K(A)) of bisphenol S with trypsin were 7.42 × 10(4) (298 K) and 5.91 × 10(4) L/mol (310 K), and those of pepsin were 5.78 × 10(4) (298 K) and 4.44 × 10(4) L/mol (310 K). Moreover, there was one main kind of binding site for bisphenol S on trypsin or pepsin.

  4. Milk Oligosaccharides Inhibit Human Rotavirus Infectivity in MA104 Cells.

    Science.gov (United States)

    Laucirica, Daniel R; Triantis, Vassilis; Schoemaker, Ruud; Estes, Mary K; Ramani, Sasirekha

    2017-09-01

    Background: Oligosaccharides in milk act as soluble decoy receptors and prevent pathogen adhesion to the infant gut. Milk oligosaccharides reduce infectivity of a porcine rotavirus strain; however, the effects on human rotaviruses are less well understood. Objective: In this study, we determined the effect of specific and abundant milk oligosaccharides on the infectivity of 2 globally dominant human rotavirus strains. Methods: Four milk oligosaccharides-2'-fucosyllactose (2'FL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and galacto-oligosaccharides-were tested for their effects on the infectivity of human rotaviruses G1P[8] and G2P[4] through fluorescent focus assays on African green monkey kidney epithelial cells (MA104 cells). Oligosaccharides were added at different time points in the infectivity assays. Infections in the absence of oligosaccharides served as controls. Results: When compared with infections in the absence of glycans, all oligosaccharides substantially reduced the infectivity of both human rotavirus strains in vitro; however, virus strain-specific differences in effects were observed. Compared with control infections, the maximum reduction in G1P[8] infectivity was seen with 2'FL when added after the onset of infection (62% reduction, P rotaviruses in MA104 cells, primarily through an effect on the virus. Although breastfed infants are directly protected, the addition of specific oligosaccharides to infant formula may confer these benefits to formula-fed infants. © 2017 American Society for Nutrition.

  5. The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

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    Laura Martinez-Alvarez

    2013-06-01

    Full Text Available A hallmark of group/species A rotavirus (RVA replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1 is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV. NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

  6. Interaction of gallic acid with trypsin analyzed by spectroscopy

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    Hao Song

    2015-06-01

    Full Text Available The interactions between trypsin and gallic acid (GA were investigated by means of fluorescence spectroscopy, UV-vis absorption spectroscopy, resonance light scattering (RLS spectroscopy, synchronous fluorescence spectroscopy, and enzymatic inhibition assay. It was found that GA can cause the fluorescence quenching of trypsin during the process of formation of GA-trypsin complex, resulting in inhibition of trypsin activity (IC50 = 3.9 × 10−6 mol/L. The fluorescence spectroscopic data showed that the quenching efficiency can reach about 80%. The binding constants were 1.9371 × 104 L/mol, 1.8192 × 104 L/mol, and 1.7465 × 104 L/mol at three temperatures, respectively. The thermodynamic parameters revealed that hydrogen bonds, van der Waals, hydrophobic, and electrostatic interactions were involved in the binding process of GA to trypsin. Molecular modeling studies illustrated a specific display of binding information and explained most of the experiment phenomena. The microenvironments of tryptophan and tyrosine residue in trypsin were changed by the GA. Results indicated that GA was a strong quencher and inhibitor of trypsin.

  7. Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

    International Nuclear Information System (INIS)

    Larsen, Anett K.; Seternes, Ole-Morten; Larsen, Merethe; Aasmoe, Lisbeth; Bang, Berit

    2008-01-01

    In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-κB) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-induced cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-κB-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts

  8. Smart PEGylation of trypsin.

    Science.gov (United States)

    Zarafshani, Zoya; Obata, Toshihiro; Lutz, Jean-François

    2010-08-09

    Thermoresponsive oligo(ethylene glycol)-based copolymers were investigated for trypsin conjugation. These copolymers have been synthesized by atom transfer radical polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO(2)MA) with oligo(ethylene glycol) methyl ether methacrylate (OEGMA(475), M(n) = 475 g.mol(-1)) at 60 degrees C in the presence of copper(I) chloride and 2,2'-bipyridyl. Two different ATRP initiators, containing succinimidyl ester moieties, were tested, namely, N-succinimidyl-2-bromopropionate and N-succinimidyl-2-bromoisobutyrate. In both cases, ATRP afforded well-defined polymers with a narrow molecular weight distribution and controlled chain-ends. However, the efficiency of initiation of the two initiators was lower than 1 and therefore the formed polymers exhibited a higher than expected mean degree of polymerization. Nevertheless, all types of polymers could be conjugated to trypsin. The conjugation reaction was performed in borax-HCl buffer. Sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. However, (co)polymers initiated by N-succinimidyl-2-bromopropionate led to the best conjugation results. The formed P(MEO(2)MA-co-OEGMA(475))-trypsin conjugates were found to be thermoresponsive and moreover exhibited a higher enzymatic activity than unmodified trypsin.

  9. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

    International Nuclear Information System (INIS)

    Kamen, B.A.; Wang, M.T.; Streckfuss, A.J.; Peryea, X.; Anderson, R.G.

    1988-01-01

    MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[ 3 H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[ 3 H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface

  10. Influence of Different Genotypes on Trypsin Inhibitor Levels and Activity in Soybeans

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    Viktor A. Nedovic

    2007-01-01

    Full Text Available This study describes the relationship between the two major trypsin inhibitors (TI in soybean, i.e., the Kunitz (KTI and Bowman-Birk (BBI trypsin inhibitors, as well as between them and the corresponding trypsin inhibitor activity (TIA. Twelve investigated soybean genotypes showed significant differences in TI levels and TIA. A very strong positive correlation was found between the levels of KTI and total BBI (r = 0.94, P < 0.05. No relationship was found between KTI, BBI or total TI and TIA. Based on this data, it appears that the levels of major TI in soybean are related. Understanding the relationship between trypsin inhibitors and their activities could be useful for further improvement of the health impacts of soy proteins.

  11. Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

    Science.gov (United States)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

  12. Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum)

    International Nuclear Information System (INIS)

    Sharma, Urvashi; Suresh, C. G.

    2011-01-01

    The purification, characterization and crystallization of a trypsin inhibitor protein isolated from chickpea seeds are reported. A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS–PAGE. The IC 50 value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2 1 2 1 2 form A, P2 1 2 1 2 form B and P2 1 2 1 2 1 . The crystals of P2 1 2 1 2 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å 3 Da −1 , corresponding to a solvent content of 42%. The other two crystal forms (P2 1 2 1 2 form B and P2 1 2 1 2 1 ) diffracted comparatively poorly

  13. Label-free electrical determination of trypsin activity by a silicon-on-insulator based thin film resistor.

    Science.gov (United States)

    Neff, Petra A; Serr, Andreas; Wunderlich, Bernhard K; Bausch, Andreas R

    2007-10-08

    A silicon-on-insulator (SOI) based thin film resistor is employed for the label-free determination of enzymatic activity. We demonstrate that enzymes, which cleave biological polyelectrolyte substrates, can be detected by the sensor. As an application, we consider the serine endopeptidase trypsin, which cleaves poly-L-lysine (PLL). We show that PLL adsorbs quasi-irreversibly to the sensor and is digested by trypsin directly at the sensor surface. The created PLL fragments are released into the bulk solution due to kinetic reasons. This results in a measurable change of the surface potential allowing for the determination of trypsin concentrations down to 50 ng mL(-1). Chymotrypsin is a similar endopeptidase with a different specificity, which cleaves PLL with a lower efficiency as compared to trypsin. The activity of trypsin is analyzed quantitatively employing a kinetic model for enzyme-catalyzed surface reactions. Moreover, we have demonstrated the specific inactivation of trypsin by a serine protease inhibitor, which covalently binds to the active site of the enzyme.

  14. Cold adaptation, ca2+ dependency and autolytic stability are related features in a highly active cold-adapted trypsin resistant to autoproteolysis engineered for biotechnological applications.

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    Alvaro Olivera-Nappa

    Full Text Available Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(° C and at least 50% more active than pig trypsin up to 50(° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.

  15. Aggregation of trypsin and trypsin inhibitor by Al cation.

    Science.gov (United States)

    Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

    2017-04-01

    Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerative diseases. We report the effect of Al cation on the solution structures of trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis, UV-Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomic force microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin into larger or more robust aggregates than trypsin inhibitor, with trypsin 5±1 SE (n=52) proteins per aggregate and for trypsin inhibitor 8.3±0.7 SE (n=118). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    Science.gov (United States)

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  17. Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

    Science.gov (United States)

    Kumari, Rakhi; Gupta, Subodh; Singh, Arvind R; Ferosekhan, S; Kothari, Dushyant C; Pal, Asim Kumar; Jadhao, Sanjay Balkrishna

    2013-01-01

    Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare

  18. Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

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    Rakhi Kumari

    Full Text Available Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04% nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02% along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio, organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01 nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate and dehydrogenases (lactate and malate, serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan

  19. The Role of Bystander Effects in the Antitumor Activity of the Hypoxia-Activated Prodrug PR-104

    Science.gov (United States)

    Foehrenbacher, Annika; Patel, Kashyap; Abbattista, Maria R.; Guise, Chris P.; Secomb, Timothy W.; Wilson, William R.; Hicks, Kevin O.

    2013-01-01

    Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such “bystander effects” may offset spatial heterogeneity in prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for PR-104, a phosphate ester pre-prodrug that is converted systemically to the hypoxia-activated prodrug PR-104A. Using Green’s function methods we calculated concentrations of oxygen, PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional tumor microregion. Model parameters were determined in vitro, using single cell suspensions to determine relationships between PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell suspensions and MCLs to PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of PR-104 activity in SiHa and HCT116 tumors, respectively. Testing the model by modulating PR-104A-activating reductases and hypoxia in tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in hypoxia and reductase expression in tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer prodrugs and could be used to identify strategies for rational prodrug optimization. PMID

  20. Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum).

    Science.gov (United States)

    Sharma, Urvashi; Suresh, C G

    2011-06-01

    A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS-PAGE. The IC(50) value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2(1)2(1)2 form A, P2(1)2(1)2 form B and P2(1)2(1)2(1). The crystals of P2(1)2(1)2 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å(3) Da(-1), corresponding to a solvent content of 42%. The other two crystal forms (P2(1)2(1)2 form B and P2(1)2(1)2(1)) diffracted comparatively poorly.

  1. Amino Acid Composition, Urease Activity and Trypsin Inhibitor Activity after Toasting of Soybean in Thick and Thin Layer

    OpenAIRE

    Krička, Tajana; Jurišić, Vanja; Voća, Neven; Ćurić, Duška; Brlek Savić, Tea; Matin, Ana

    2009-01-01

    The objective of this study was to determine amino acid content, urease activity and trypsin inhibitor activity in soybean grain for polygastric animals’ feed aft er toasting with the aim to introduce thick layer in toasting technology. Hence, soybean was toasted both in thick and thin layer at 130 oC during 10 minutes. In order to properly monitor the technological process of soybean thermal processing, it was necessary to study crude protein content, urease activity, trypsin inhibitor activ...

  2. Influence of surface-imprinted nanoparticles on trypsin activity.

    Science.gov (United States)

    Guerreiro, António; Poma, Alessandro; Karim, Kal; Moczko, Ewa; Takarada, Jessica; de Vargas-Sansalvador, Isabel Perez; Turner, Nicholas; Piletska, Elena; de Magalhães, Cristiana Schmidt; Glazova, Natalia; Serkova, Anastasia; Omelianova, Aleksandra; Piletsky, Sergey

    2014-09-01

    Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

    Science.gov (United States)

    Samac, Deborah; Storey, Richard

    1981-01-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

  4. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  5. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  6. Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

    Science.gov (United States)

    Zamani, Abbas; Benjakul, Soottawat

    2016-02-01

    Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

  7. Trypsinization and the radiosensitivity of mitotic and log phase Chinese hamster V79 cells exposed to 250 kVp X-rays

    International Nuclear Information System (INIS)

    Reddy, N.M.S.; Stevenson, A.F.G.; Lange, C.S.

    1989-01-01

    The authors studied the influence of trypsin-induced morphological changes on the x-radiosensitivity of cells plated at either low (4-600/cm 2 ) or high (2 x 10 4 /cm 2 ) density and grown overnight before treatments. Trypsin treatment induced contraction and rounding of spread cells. The results suggest that: (1) trypsin-induced cell contraction affects the ability of cells to repair radiation damage, (2) spread cells are better able to repair potential lethal damage (PLD) than rounded cells, (3) immediate plating survival of cells in high-density cultures may not represent their intrinsic radiosensitivity and (4) cell-to-cell contact is not necessary for log phase cells to repair PLD. (author)

  8. Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix).

    Science.gov (United States)

    Klomklao, Sappasith; Benjakul, Soottawat; Visessanguan, Wonnop; Kishimura, Hideki; Simpson, Benjamin K

    2007-12-01

    Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 degrees C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 degrees C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl2 partially protected trypsin against activity loss at 40 degrees C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.

  9. Radiobiological studies of cells in multicellular spheroids using a sequential trypsinization technique

    International Nuclear Information System (INIS)

    Giesbrecht, J.L.; Wilson, W.R.; Hill, R.P.

    1981-01-01

    The radiation response of V79 Chinese hamster cells grown as multicellular spheroids has been investigated by determining survival curves for treatment under a variety of different oxygen concentrations. Spheroids were irradiated under fully oxygenated conditions in air-equilibrated medium at 37 0 C, in medium exposed to lower oxygen tension (5% O 2 ) for times varying from 1 hr to 3 days, or under anoxic conditions. For comparison with the spheroids, using identical treatment conditions, V79 cells were grown in suspension as a subconfluent monolayer attached to Sephadex (microcarrier) beads and irradiated under fully oxygenated or anoxic conditions. The radiation response of cells at different depths within the spheroid was investigated by using a sequential trypsinization technique developed to remove eight or nine shells of cells successively from the spheroid surface. When irradiation was given under fully oxygenated conditions the outer few cell layers were more sensitive than the inner cells, a finding which is not understood. As expected the inner cells in spheroids irradiated in air (at 37 0 C) or in 5% O 2 are more resistant than the outer cells. For an acute exposure to 5% O 2 (1 hr) in the inner cells displayed full radiobiological hypoxia; however, for chronic exposures to low oxygen this was not the case. These results with the sequential trypsinization procedure suggest that the radiation response of cells in spheroids is more complex than anticipted

  10. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  11. Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

    Science.gov (United States)

    XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

    2015-01-01

    The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

  12. Effects of tannic acid on trypsin and leucine aminopeptidase activities in gypsy moth larval midgut

    Directory of Open Access Journals (Sweden)

    Mrdaković Marija

    2013-01-01

    Full Text Available The effects of allelochemical stress on genetic variations in the specific activities of gypsy moth digestive enzymes (trypsin and leucine aminopeptidase and relative midgut mass (indirect measure of food consumption, as well as variability in their plasticity, were investigated in fifth instar gypsy moths originating from two populations with different trophic adaptations (oak and locust-tree forests. Thirty-two full-sib families from the Quercus population and twenty-six full-sib families from the Robinia population were reared on an artificial diet with or without supplementation with tannic acid. Between population differences were observed as higher average specific activity of trypsin and relative midgut mass in larvae from the Robinia population. Significant broad-sense heritabilities were observed for the specific activity of trypsin in the control state, and for specific activity of leucine aminopeptidase in a stressful environment. Significantly lower heritability for relative midgut mass was recorded in larvae from the Robinia population reared under stressful conditions. Significant variability of trypsin plasticity in larvae from both populations and significant variability of leucine aminopeptidase plasticity in larvae from the Robinia population point to the potential for the evolution of enzyme adaptive plastic responses to the presence of stressor. Non-significant across-environment genetic correlations do not represent a constraint for the evolution of enzyme plasticity. [Projekat Ministarstva nauke Republike Srbije, br. 173027

  13. A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

    Science.gov (United States)

    Fang, Evandro Fei; Ng, Tzi Bun

    2015-04-01

    Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.

  14. Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

    Science.gov (United States)

    Cruz, Ana C B; Massena, Fábio S; Migliolo, Ludovico; Macedo, Leonardo L P; Monteiro, Norberto K V; Oliveira, Adeliana S; Macedo, Francisco P; Uchoa, Adriana F; Grossi de Sá, Maria F; Vasconcelos, Ilka M; Murad, Andre M; Franco, Octavio L; Santos, Elizeu A

    2013-09-01

    The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  15. New active analogues of Cucurbita maxima trypsin inhibitor III (CMTI-III) modified in the non-contact region.

    Science.gov (United States)

    Rózycki, J; Kupryszewski, G; Rolka, K; Ragnarsson, U; Zbyryt, T; Krokoszyńska, I; Wilusz, T

    1994-01-01

    Four new analogues of trypsin inhibitor CMTI-III(3-28) = [desArg1,desVal2,desGly29]CMTI-III which was recently shown to be fully active, were synthesized by the solid-phase method. The introduction of glycine in position 9 (peptide 1) and Gly-Pro-Gly (peptide 2) and Gly-Pro-Asn (peptide 3) in the regions 17-19 and 23-25, respectively, did not change the antitrypsin activity of all modified peptides. All of these substitutions are presumed to be outside the trypsin-binding loop as judged from the X-ray structure of the complex between beta-trypsin and the related inhibitor CMTI-I. Also the fourth analogue which was substituted in all the positions mentioned, exhibited the full activity.

  16. Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

    Science.gov (United States)

    Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

    2015-05-01

    A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  17. A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds.

    Science.gov (United States)

    Krishnamoorthi, R; Gong, Y X; Richardson, M

    1990-10-29

    A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.

  18. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    Directory of Open Access Journals (Sweden)

    M. F. CUSTÓDIO

    2009-01-01

    Full Text Available

    This work presents a method for adding value to cheese whey residues by whey proteins hydrolysis, using trypsin, chymotrypsin and carboxypeptidase A as catalysts. Sweet cheese whey was dialyzed and filtered in kaolin. Lactose and protein contents were analyzed after each step. The activities of bovine pancreas trypsin and chymotrypsin were measured at different pHs and temperatures. The optimal pH for the hydrolysis of whey proteins was 9.0 for both enzymes. Optima temperatures were 60ºC for trypsin, and 50ºC for chymotrypsin. Trypsin exhibited typical Michaelis-Menten behavior, but chymotrypsin did not. Electrophoretic analysis showed that neither trypsin nor chymotrypsin alone hydrolyzed whey proteins in less than three hours. Hydrolysis rates of -lactalbumin by trypsin, and of bovine serum albumin by chymotrypsin were low. When these enzymes were combined, however, all protein fractions were attacked and rates of hydrolysis were enhanced by one order of magnitude. The addition of carboxypeptidase A to the others enzymes did not improve the process yield.

  19. Lectins as carriers: preparation and purification of a concanavalin A-trypsin conjugate

    International Nuclear Information System (INIS)

    Shier, W.T.

    1985-01-01

    The scheme for the preparation and purification of Con A-trypsin demonstrates the presence of Con A in the conjugate by affinity purification and by hemagglutination. The presence of Con A was demonstrated in two additional ways. The conjugate was prepared using trypsin labeled with iodine 125 and unlabeled Con A. The resulting conjugate containing 42,800 cpm/mg protein was used to demonstrate prolonged retention of the conjugated trypsin in mouse footpads. The presence of trypsin was also demonstrated in the conjugate by affinity chromatography and by the esterase activity characteristic of trypsin with tosyl-L-arginine methyl ester as substrate. Con A-trypsin was also assayed for proteolytic activity with a macromolecular substrate azocasein. A comparison of proteolytic activities with these two substrates indicated that 50-80% of the proteolytic activity observed with the small substrate is retained with macromolecular substrates

  20. Laura: Soybean variety lacking Kunitz trypsin inhibitor

    Directory of Open Access Journals (Sweden)

    Srebrić Mirjana

    2010-01-01

    Full Text Available Grain of conventional soybean varieties requires heat processing to break down trypsin inhibitor's activity before using as food or animal feed. At the same time, protein denaturation and other qualitative changes occur in soybean grain, especially if the temperature of heating is not controlled. Two types of trypsin inhibitor were found in soybean grain the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor. Mature grain of soybean Laura is lacking Kunitz trypsin inhibitor. Grain yield of variety Laura is equal to high yielding varieties from the maturity group I, where it belongs. Lacking of Kunitz-trypsin inhibitor makes soybean grain suitable for direct feeding in adult non ruminant animals without previous thermal processing. Grain of variety Laura can be processed for a shorter period of time than conventional soybeans. This way we save energy, and preserve valuable nutritional composition of soybean grain, which is of interest in industrial processing.

  1. Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

    International Nuclear Information System (INIS)

    Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

    2010-01-01

    Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K i , 0.1 x 10 -9 M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.

  2. Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

    2009-04-01

    A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

  3. Trypsin radioimmunoassay

    International Nuclear Information System (INIS)

    Scheithauer, R.; Wolf, F.; Tympner, F.

    1981-01-01

    In 29 patients with suspicion of pancreatic disease standard secretin-pancreozymin-test was performed parallel to trypsin determination by radioimmunoassay before and after stimulation with secretin. Mean serum trypsin in normal subjects was 175 ng BIT/ml (range 90-250), the maximum after stimulation being 20 minutes after secreting injection (range 110-550). Preliminary normal values are 270 ng BIT/ml for basal concentration and 650 ng BIT/ml for 20 min after secretin stimulation. In the group of normals there was no case of misinterpretation. In patients with several pathological parameters (n = 10) basal trypsin concentration was increased in 7 cases, the stimulated value was concordant with the definite diagnosis in every case. Significant advantage for diagnostics was derived in patients having had pancreatic diseases before, however being actually normal with respect to standard diagnostic parameters. All these patients revealed increased trypsin concentrations after stimulation and 50% of them showed increased basal values. Equivocal results were seen in patients with endstage pancreatitis as well as in case of obstruction during reduced secretion. (orig.) [de

  4. Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Evandro Fei; Wong, Jack Ho [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR (China); Bah, Clara Shui Fern [Department of Food Science, Division of Sciences, University of Otago (New Zealand); Lin, Peng [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR (China); Tsao, Sai Wah [Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Sassoon Road, Pokfulam, Hong Kong SAR (China); Ng, Tzi Bun, E-mail: b021770@mailserv.cuhk.edu.hk [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR (China)

    2010-06-11

    Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.

  5. Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

    Science.gov (United States)

    Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

    2010-06-11

    Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. (c) 2010 Elsevier Inc. All rights reserved.

  6. Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway

    Directory of Open Access Journals (Sweden)

    So EC

    2014-02-01

    Full Text Available Edmund Cheung So,1,2 Yu-Xuan Lin,3 Chi Hao Tseng,1 Bo-Syong Pan,3 Ka-Shun Cheng,2 Kar-Lok Wong,2 Lyh-Jyh Hao,4 Yang-Kao Wang,5 Bu-Miin Huang2 1Department of Anesthesia, Tainan Municipal An Nan Hospital, China Medical University, Tainan, Taiwan; 2Department of Anesthesia, China Medical University, Taichung, Taiwan; 3Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan, Taiwan; 4Department of Internal Medicine, Division of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch Tainan, Taiwan; 5Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan Purpose: The present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells. Methods: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods. Results: Data showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase. Conclusion: Midazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways. Keywords: midazolam, apoptosis, MA-10 cell, caspase, Akt, MAPKs

  7. 42 CFR 438.104 - Marketing activities.

    Science.gov (United States)

    2010-10-01

    ... State government, or similar entity. (c) State agency review. In reviewing the marketing materials... 42 Public Health 4 2010-10-01 2010-10-01 false Marketing activities. 438.104 Section 438.104... (CONTINUED) MEDICAL ASSISTANCE PROGRAMS MANAGED CARE Enrollee Rights and Protections § 438.104 Marketing...

  8. Orthosteric and Allosteric Regulation in Trypsin-Like Peptidases

    DEFF Research Database (Denmark)

    Kromann-Tofting, Tobias

    Trypsin-like serine peptidases play an important role in many physiological and pathophysiological processes, the latter including cardiovascular diseases and cancer. Binding of natural ligands to functional sites on the peptidase surface balances the level of activity and substrate specificity......-ray crystallography to determine crystal structures of active and inactive conformations of muPA, combined with biochemical analysis, elucidated an allosteric regulatory mechanism, which is now believed to be highly conserved in the trypsin-like serine peptidases. Targeting zymogen activation represents an attractive...

  9. Trypsin Binding with Copper Ions Scavenges Superoxide: Molecular Dynamics-Based Mechanism Investigation

    Directory of Open Access Journals (Sweden)

    Xin Li

    2018-01-01

    Full Text Available Trypsin is a serine protease, which has been proved to be a novel superoxide scavenger. The burst of superoxide induced by polychlorinated biphenyls can be impeded by trypsin in both wild type and sod knockout mutants of Escherichia coli. The experimental results demonstrated that the activities of superoxide scavenging of trypsin were significantly accelerated by Cu ions. Also, with the addition of Cu ions, a new β-sheet (β7 transited from a random coil in the Cu(II-trypsin (TP system, which was favorable for the formation of more contacts with other sheets of trypsin. Residue–residue network analysis and the porcupine plots proved that the Cu ion in trypsin strengthened some native interactions among residues, which ultimately resulted in much greater stability of the Cu(II-TP system. Moreover, compact and stable trypsin structures with Cu ions might be responsible for significantly provoking the activity of superoxide scavenging.

  10. A novel protoapigenone analog RY10-4 induces breast cancer MCF-7 cell death through autophagy via the Akt/mTOR pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xuenong; Wei, Han; Liu, Ziwei; Yuan, Qianying [Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Wei, Anhua [Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Shi, Du; Yang, Xian [Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Ruan, Jinlan, E-mail: jinlan8152@163.com [Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China)

    2013-07-15

    Protoapigenone is a unique flavonoid and enriched in many ferns, showing potent antitumor activity against a broad spectrum of human cancer cell lines. RY10-4, a modified version of protoapigenone, manifested better anti-proliferation activity in human breast cancer cell line MCF-7. The cytotoxicity of RY10-4 against MCF-7 cells is exhibited in both time- and concentration-dependent manners. Here we investigated a novel effect of RY10-4 mediated autophagy in autophagy defect MCF-7 cells. Employing immunofluorescence assay for microtubule-associated protein light-chain 3 (LC3), monodansylcadaverine staining, Western blotting analyses for LC3 and p62 as well as ultrastructural analysis by transmission electron microscopy, we showed that RY10-4 induced autophagy in MCF-7 cells but protoapigenone did not. Meanwhile, inhibition of autophagy by pharmacological and genetic approaches significantly increased the viability of RY10-4 treated cells, suggesting that the autophagy induced by RY10-4 played as a promotion mechanism for cell death. Further studies revealed that RY10-4 suppressed the activation of mTOR and p70S6K via the Akt/mTOR pathway. Our results provided new insights for the mechanism of RY10-4 induced cell death and the cause of RY10-4 showing better antitumor activity than protoapigenone, and supported further evidences for RY10-4 as a lead to design a promising antitumor agent. - Highlights: • We showed that RY10-4 induced autophagy in MCF-7 cells but protoapigenone did not. • Autophagy induced by RY10-4 played as a promotion mechanism for cell death. • RY10-4 induced autophagy in MCF-7 cell through the Akt/mTOR pathway. • We provided new insights for the mechanism of RY10-4 induced cell death.

  11. Chitosan nanoparticles-trypsin interactions: Bio-physicochemical and molecular dynamics simulation studies.

    Science.gov (United States)

    Salar, Safoura; Mehrnejad, Faramarz; Sajedi, Reza H; Arough, Javad Mohammadnejad

    2017-10-01

    Herein, we investigated the effect of the chitosan nanoparticles (CsNP) on the structure, dynamics, and activity of trypsin. The enzyme activity in complex with the nanoparticles slightly increased, which represents the interactions between the nanoparticles and the enzyme. The kinetic parameters of the enzyme, K m and k cat , increased after adding the nanoparticles, resulting in a slight increase in the catalytic efficiency (k cat /K m ). However, the effect of the nanoparticles on the kinetic stability of trypsin has not exhibited significant variations. Fluorescence spectroscopy did not show remarkable changes in the trypsin conformation in the presence of the nanoparticles. The circular dichroism (CD) spectroscopy results also revealed the secondary structure of trypsin attached to the nanoparticles slightly changed. Furthermore, we used molecular dynamics (MD) simulation to find more information about the interaction mechanisms between the nanoparticles and trypsin. The root mean square deviation (RMSD) of Cα atoms results have shown that in the presence of the nanoparticles, trypsin was stable. The simulation and the calculation of the binding free energy demonstrate that the nonpolar interactions are the most important forces for the formation of stable nanoparticle-trypsin complex. This study has explicitly elucidated that the nanoparticles have not considerable effect on the trypsin. Copyright © 2017. Published by Elsevier B.V.

  12. The trypsin-catalyzed hydrolysis of monomolecular films of lysylphosphatidylglycerol

    NARCIS (Netherlands)

    Gould, R.M.; Dawson, R.M.C.

    1972-01-01

    The hydrolysis by trypsin of the bacterial phospholipid, lysylphosphatidyl-glycerol has been studied at the air-water interface. High specific activity [14C]-lysylphosphatidylglycerol was prepared biosynthetically and the trypsin action followed by measuring the loss of surface radioactivity from a

  13. The kinetics of interaction of porcine - alpha-, and porcine - beta -trypsin with intact and modified soybean trypsin inhibitor (kunitz)

    International Nuclear Information System (INIS)

    Hamid, M.A.

    1994-01-01

    The association of porcine trypsin with soybean trypsin inhibitor (Kunitz) resulted in characteristic changes in absorption spectrum, indicating an alteration of the micro environments of the enzyme chromophores as a consequence of the interaction. The rates of formation of the stable trypsin - inhibitor complexes from porcine - alpha - trypsin and soybean trypsin inhibitor and from porcine - beta - trypsin and either intact or modified soybean trypsin inhibitor were measured by mixing the equimolar concentration of the reactants in a Stopped - Flow apparatus at pH (4.5 to 10.0). The reaction of trypsin with soybean trypsin inhibitor was of first order with respect to the concentration of the reactants used. The rates of dissociation of the stable complexes, alpha - trypsin - soybean trypsin inhibitor, beta -trypsin - soybean trypsin inhibitor and beta -trypsin modified soybean trypsin inhibitor were also measured at pH (1.92 to 3.58). The values of first order rate constant, k/sub D/ obtained for the dissociation of all the three complexes were identical with one another. The kinetics results obtained for the porcine trypsin were compared with those of bovine trypsin system and it was suggested that the reaction mechanisms in both these systems were identical. (author)

  14. Inhibition of trypsin by condensed tannins and wine.

    Science.gov (United States)

    Gonçalves, Rui; Soares, Susana; Mateus, Nuno; de Freitas, Victor

    2007-09-05

    Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.

  15. Comparison of mesenchymal stem cells released from poly(N-isopropylacrylamide) copolymer film and by trypsinization

    International Nuclear Information System (INIS)

    Yang Lei; Liu Tianqing; Song Kedong; Jiang Lili; Wu Shuang; Guo Wenhua; Cheng Fang; Lu, Jian R

    2012-01-01

    Temperature-responsive platforms containing poly(N-isopropylacrylamide) (PNIPAAm) have been developed as an effective substitute for enzymatic treatment to recover adherent cells, but it remains unclear whether this alternative harvesting method tends to support stem cells preserving them being primitive. This study mainly investigated the biological properties of mesenchymal stem cells derived from rat bone marrow and human adipose tissue (BM-MSCs and AT-MSCs) after being cultured on PNIPAAm copolymer films and recovered by temperature drop, and compared the cells harvested from glass coverslips with trypsinization as controls. The experimental results demonstrated that after three serial passages, the released MSCs from thermal liftoff showed no significant differences in cell morphology, immunophenotype and osteogenesis for BM-MSCs or adipogenesis for AT-MSCs, but had higher viability, stronger proliferation and higher adipogenic differentiation for BM-MSCs or higher osteogenic differentiation for AT-MSCs compared with the trypsinization group. Besides, more proteins remained around or within the cell membranes upon temperature drop. It is concluded that cell detachment with more extracellular matrix proteins facilitates the maintenance of membrane proteins, and accordingly preserves MSC properties related to viability, proliferation and differentiation to some extent. This indicates that the PNIPAAm copolymer films and their matching cooling treatment can be used as effective alternatives to the existing culture substrates and traditional enzymatic digestion for MSCs. (paper)

  16. Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Hinterwirth, Helmut; Lindner, Wolfgang [Department of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, 1090 Vienna (Austria); Laemmerhofer, Michael, E-mail: michael.laemmerhofer@uni-tuebingen.de [Department of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, 1090 Vienna (Austria)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Size and spacer affect bioactivity of nanoparticulate trypsin reactor. Black-Right-Pointing-Pointer Increase of GNP's size increases activity of bound trypsin. Black-Right-Pointing-Pointer Increase of spacer length increases amount and activity of immobilized enzyme by factor 6. Black-Right-Pointing-Pointer Decrease of digestion time up to less than 1 h when trypsin immobilized onto GNPs. Black-Right-Pointing-Pointer Reduced auto-digestion compared to trypsin in-solution. - Abstract: The systematic study of activity, long-time stability and auto-digestion of trypsin immobilized onto gold nanoparticles (GNPs) is described in this paper and compared to trypsin in-solution. Thereby, the influence of GNP's size and immobilization chemistry by various linkers differing in lipophilicity/hydrophilicity and spacer lengths was investigated with regard to the bioactivity of the conjugated enzyme. GNPs with different sizes were prepared by reduction and simultaneous stabilization with trisodium citrate and characterized by UV/vis spectra, dynamic light scattering (DLS), {zeta}-potential measurements and transmission electron microscopy (TEM). GNPs were derivatized by self-assembling of bifunctional thiol reagents on the nanoparticle (NP) surface via dative thiol-gold bond yielding a carboxylic acid functionalized surface. Trypsin was either attached directly via hydrophobic and ionic interactions onto the citrate stabilized GNPs or immobilized via EDC/NHS bioconjugation onto the carboxylic functionalized GNPs, respectively. The amount of bound trypsin was quantified by measuring the absorbance at 280 nm. The activity of bound enzyme and its Michaelis Menten kinetic parameter K{sub m} and v{sub max} were measured by the standard chromogenic substrate N{sub {alpha}}-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA). Finally, digestion of a standard protein mixture with the trypsin-conjugated NPs followed by analysis with

  17. Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity

    International Nuclear Information System (INIS)

    Hinterwirth, Helmut; Lindner, Wolfgang; Lämmerhofer, Michael

    2012-01-01

    Highlights: ► Size and spacer affect bioactivity of nanoparticulate trypsin reactor. ► Increase of GNP's size increases activity of bound trypsin. ► Increase of spacer length increases amount and activity of immobilized enzyme by factor 6. ► Decrease of digestion time up to less than 1 h when trypsin immobilized onto GNPs. ► Reduced auto-digestion compared to trypsin in-solution. - Abstract: The systematic study of activity, long-time stability and auto-digestion of trypsin immobilized onto gold nanoparticles (GNPs) is described in this paper and compared to trypsin in-solution. Thereby, the influence of GNP's size and immobilization chemistry by various linkers differing in lipophilicity/hydrophilicity and spacer lengths was investigated with regard to the bioactivity of the conjugated enzyme. GNPs with different sizes were prepared by reduction and simultaneous stabilization with trisodium citrate and characterized by UV/vis spectra, dynamic light scattering (DLS), ζ-potential measurements and transmission electron microscopy (TEM). GNPs were derivatized by self-assembling of bifunctional thiol reagents on the nanoparticle (NP) surface via dative thiol-gold bond yielding a carboxylic acid functionalized surface. Trypsin was either attached directly via hydrophobic and ionic interactions onto the citrate stabilized GNPs or immobilized via EDC/NHS bioconjugation onto the carboxylic functionalized GNPs, respectively. The amount of bound trypsin was quantified by measuring the absorbance at 280 nm. The activity of bound enzyme and its Michaelis Menten kinetic parameter K m and v max were measured by the standard chromogenic substrate N α -Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA). Finally, digestion of a standard protein mixture with the trypsin-conjugated NPs followed by analysis with LC–ESI-MS and successful MASCOT search demonstrated the applicability of the new heterogenous nano-structured biocatalyst. It could be shown that the

  18. Prior lactose glycation of caseinate via the Maillard reaction affects in vitro activities of the pepsin-trypsin digest toward intestinal epithelial cells.

    Science.gov (United States)

    Wang, X P; Zhao, X H

    2017-07-01

    The well-known Maillard reaction in milk occurs between lactose and milk proteins during thermal treatment, and its effects on milk nutrition and safety have been well studied. A lactose-glycated caseinate was prepared via this reaction and digested using 2 digestive proteases, pepsin and trypsin. The glycated caseinate digest was assessed for its in vitro activities on rat intestinal epithelial cells in terms of growth proliferation, anti-apoptotic effect, and differentiation induction using caseinate digest as reference, to verify potential effects of the Maillard reaction on these activities of caseinate digest to the cells. Two digests had proliferative and anti-apoptotic effects, and reached the highest effects at 0.02 g/L of digest concentration with treatment time of 24 h. In comparison with caseinate digest, glycated caseinate digest always showed weaker proliferative (5.3-14.2%) and anti-apoptotic (5.9-39.0%) effects, and was more toxic to the cells at 0.5 g/L of digest concentration with treatment time of 48 h. However, glycated caseinate digest at 2 incubation times of 4 to 7 d showed differentiation induction higher than caseinate digest, as it could confer the cells with increased activities in lactase (16.3-26.6%), sucrase (22.4-31.2%), and alkaline phosphatase (17.4-24.8%). Transmission electron microscopy observation results also confirmed higher differentiation induction of glycated caseinate digest. Amino acid loss and lactose glycation partially contributed to these decreased and enhanced activities of glycated caseinate digest, respectively. The Maillard reaction of caseinate and lactose is thus shown in this study to have effects on the activities of caseinate digest to intestinal epithelial cells. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  19. Trypsin inhibitory activity of artemisinin and its biotransformed product

    International Nuclear Information System (INIS)

    Shahwar, D.; Raza, M.A.

    2013-01-01

    Summary: Artemisinin (1 ), a sesquiterpene lactone is an important constituent of anti-malarial drugs. In the present study, it was extracted from aerial parts of Artemisia roxburghiana Besser. Biotransformation of artemisinin ( 1 ) was carried out in the culture of Aspergillus niger GC-4 which yielded 5-hydroxy artemisinin (2 ) The structures of 1-2 were confirmed through spectral studies. Both compounds were screened against trypsin using colorimetric method. The biotransformed product 2 showed significant protease inhibitory activity with 53.5 +- 1.6% inhibition and IC/sub 50/ = 0.29 +- 0.02 mM as compared to artemisinin (20.4 +- 0.3% inhibition). (author)

  20. Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors.

    Science.gov (United States)

    Machado, Suzy Wider; de Oliveira, Caio Fernando Ramalho; Zério, Neide Graciano; Parra, José Roberto Postali; Macedo, Maria Lígia Rodrigues

    2017-08-01

    Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs. © 2017 Wiley Periodicals, Inc.

  1. Free radical inactivation of trypsin

    International Nuclear Information System (INIS)

    Cudina, Ivana; Jovanovic, S.V.

    1988-01-01

    Reactivities of free radical oxidants, radical OH, Br2-anion radical and Cl 3 COO radical and a reductant, CO2-anion radical, with trypsin and reactive protein components were determined by pulse radiolysis of aqueous solutions at pH 7, 20 0 C. Highly reactive free radicals, radical OH, Br2-anion radical and CO2-anion radical, react with trypsin at diffusion controlled rates. Moderately reactive trichloroperoxy radical, k(Cl 3 COO radical + trypsin) preferentially oxidizes histidine residues. The efficiency of inactivation of trypsin by free radicals is inversely proportional to their reactivity. The yields of inactivation of trypsin by radical OH, Br2-anion radical and CO2-anion radical are low, G(inactivation) = 0.6-0.8, which corresponds to ∼ 10% of the initially produced radicals. In contrast, Cl 3 COO radical inactivates trypsin with ∼ 50% efficiency, i.e. G(inactivation) = 3.2. (author)

  2. Quantification of trypsin with a radioimmunoassay in herring larvae (Clupea harengus L.) compared with a highly sensitive fluorescence technique to determine tryptic enzyme activity

    International Nuclear Information System (INIS)

    Ueberschaer, B.; Pedersen, B.H.; Hjelmeland, K.

    1993-01-01

    Enzymatic activity and quantity of the protease trypsin were measured in individual herring larvae (Clupea harengus L.). The enzymatic activity assay was done by a fluorescence technique, and a radioimmunoassay was used for quantification of trypsin. The results are compared and the differences between the techniques discussed. Both methods have similar results, as high or low values in trypsin quantity were reflected in high or low values of tryptic activity. Quantity and activity were linearly and positively correlated, but small differences between methods were found at the lowest detection limits. Both techniques reflect the high variability between individual larvae. (orig.)

  3. Interaction of methotrexate with trypsin analyzed by spectroscopic and molecular modeling methods

    Science.gov (United States)

    Wang, Yanqing; Zhang, Hongmei; Cao, Jian; Zhou, Qiuhua

    2013-11-01

    Trypsin is one of important digestive enzymes that have intimate correlation with human health and illness. In this work, the interaction of trypsin with methotrexate was investigated by spectroscopic and molecular modeling methods. The results revealed that methotrexate could interact with trypsin with about one binding site. Methotrexate molecule could enter into the primary substrate-binding pocket, resulting in inhibition of trypsin activity. Furthermore, the thermodynamic analysis implied that electrostatic force, hydrogen bonding, van der Waals and hydrophobic interactions were the main interactions for stabilizing the trypsin-methotrexate system, which agreed well with the results from the molecular modeling study.

  4. Performance comparison of three trypsin columns used in liquid chromatography?

    OpenAIRE

    ?lechtov?, Tereza; Gilar, Martin; Kal?kov?, Kv?ta; Moore, Stephanie M.; Jorgenson, James W.; Tesa?ov?, Eva

    2017-01-01

    Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 ? pores, packed in 2.1 ? 30 m...

  5. The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

    Science.gov (United States)

    Rypniewski, W R; Hastrup, S; Betzel, C; Dauter, M; Dauter, Z; Papendorf, G; Branner, S; Wilson, K S

    1993-06-01

    The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

  6. Potential toxicity of phthalic acid esters plasticizer: interaction of dimethyl phthalate with trypsin in vitro.

    Science.gov (United States)

    Wang, Yaping; Zhang, Guowen; Wang, Langhong

    2015-01-14

    Dimethyl phthalate (DMP) is widely used as a plasticizer in industrial processes and has been reported to possess potential toxicity to the human body. In this study, the interaction between DMP and trypsin in vitro was investigated. The results of fluorescence, UV–vis, circular dichroism, and Fourier transform infrared spectra along with cyclic voltammetric measurements indicated that the remarkable fluorescence quenching and conformational changes of trypsin resulted from the formation of a DMP–trypsin complex, which was driven mainly by hydrophobic interactions. The molecular docking and trypsin activity assay showed that DMP primarily interacted with the catalytic triad of trypsin and led to the inhibition of trypsin activity. The dimensions of the individual trypsin molecules were found to become larger after binding with DMP by atomic force microscopy imaging. This study offers a comprehensive picture of DMP–trypsin interaction, which is expected to provide insights into the toxicological effect of DMP.

  7. The effect of bound to dialdehudecellulose protein concentration on the activity of immobilized trypsin after γ-irradiation and in process of storage

    International Nuclear Information System (INIS)

    Belov, A.A.; Ryl'tsev, V.V.; Ignatyuk, T.E.; Filatov, V.N.

    1994-01-01

    It is found the complex effect of the bound enzyme concentration on the proteolytic activity of trypsin immobilized to dialdehydecellulose (preriodate oxidation) after γ-irradiation and in process of storage. It is shown the occurance of three stages of immobilized enzyme inactivation in process of immobilization and storage. The velocity of inactivation did not depend on bound trypsin concentration. The ratio of proteolytic activity of samples before and after γ-irradiation was increased with the increase of immobilized to carrier enzyme concentration and was not change (in range of experiment error) in process of storage. The results were compared with that of cryctlline trypsin

  8. Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

    Science.gov (United States)

    Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M

    2003-06-01

    The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

  9. Screening and purification of a novel trypsin inhibitor from Prosopis juliflora seeds with activity toward pest digestive enzymes.

    Science.gov (United States)

    Sivakumar, S; Franco, O L; Tagliari, P D; Bloch, C; Mohan, M; Thayumanavan, B

    2005-08-01

    Several pests are capable of decreasing crop production causing severe economical and social losses. Aiming to find novel molecules that could impede the digestion process of different pests, a screening of alpha-amylase and trypsin-like proteinase inhibitors was carried out in Prosopis juliflora, showing the presence of both in dry seeds. Furthermore, a novel trypsin inhibitor, with molecular mass of 13,292 Da, was purified showing remarkable in vitro activity against T. castaneum and C. maculatus.

  10. Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy

    OpenAIRE

    Iu. А. Gordiienko; Ya. V. Babets; А. О. Kulinich; А. І. Shevtsova; G. О. Ushakova

    2014-01-01

    Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). ...

  11. Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco.

    Science.gov (United States)

    Ennima, Imane; Sebbar, Ghizlane; Harif, Bachir; Amzazi, Saaid; Loutfi, Chafiqa; Touil, Nadia

    2016-05-05

    Group A rotaviruses (RVA) are the main cause of neonatal calve diarrhea (NCD) in Morocco. In this study, we isolated RVA strains from NCD clinical samples in order to support RVA disease control in Morocco. This isolation process constitutes a first step toward vaccine development. Thirteen fecal samples were obtained from calves with a single episode of neonate calf diarrhea at three different dairies and two samples were collected from field during a severe NCD outbreak. Diagnosis of RVA infection was based on fecal immune-chromatographic rapid test and further evaluated for their hemagglutination (HA) activity. RVA isolation was carried out on MA104 cells after inoculates were treated with different concentrations of trypsin TPCK. All RVA isolates were confirmed by LSI VetMAX™ Triplex Ruminant Rotavirus & Coronavirus Real-Time PCR kit. G and P typing were determined by direct sequencing of the VP4 and VP7 amplicons. RVA isolation was achieved for nine clinical samples following one or two passages (60 %) and was properly depended on HA activity and trypsin treatment of inoculates. The first sign of CPE detected consisted of increased cell granularity, obscure cell boundaries, cell rounding, and eventual degeneration and detachment of cells. At lower TPCK concentration (3-10 μg/inoculum), no changes at the cellular level were observed, while cells activated with 25-30 μg of trypsin/inoculums, they degenerated and trypsin cytotoxicity was enhanced. Appreciable changes in cell's morphology were detected with optimal trypsin concentration of 15-20 μg trypsin/inoculums. Data from qRT-PCR confirmed that unsuccessful cultivations have No-Ct, and all nine isolates have Ct values ranged between 12.17 and 24.69. Analysis sequencing revealed that field isolates were of G6 P[5] serotype and isolates from the dairy NCD samples were of G10 P[14] serotype. To our knowledge, this is the first study in Morocco which reports the circulation of G10P[14] in NCD on dairy

  12. The use of poly(ethylene terephthalate)-poly(aniline) composite for trypsin immobilisation

    Energy Technology Data Exchange (ETDEWEB)

    Caramori, S.S. [Laboratorio de Quimica de Proteinas, Departamento de Bioquimica e Biologia Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Goias, Cx. Postal 131, 74001-970 Goiania-GO (Brazil)], E-mail: samanthabio@hotmail.com; Fernandes, K.F. [Laboratorio de Quimica de Proteinas, Departamento de Bioquimica e Biologia Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Goias, Cx. Postal 131, 74001-970 Goiania-GO (Brazil)], E-mail: katia@icb.ufg.br

    2008-08-01

    This paper presents trypsin immobilisation on strips of poly(ethylene terephthalate)-poly(aniline), activated with glutaraldehyde (PET-PANIG) composite. The photomicrography of the material showed changes corresponding to the chemical modifications produced in the steps of synthesis. The immobilisation process was very efficient under optimal conditions (18.6%). The immobilised and free enzyme presented the same pH and temperature optimum. PET-PANIG-trypsin was able to hydrolyse casein, albumin, gelatine, and skimmed milk. Km{sub app} value for PET-PANIG-trypsin was very close to Km of the free enzyme for casein. Immobilised trypsin showed higher stability than the free enzyme, with 100% activity after 14 days of storage at 4 deg. C and 100% operational stability after 4 cycles of use.

  13. Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor: trypsin complex

    International Nuclear Information System (INIS)

    Woodward, C.K.; Ellis, L.M.

    1975-01-01

    The hydrogen exchange kinetics of the complex of trypsin--soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins. (U.S.)

  14. TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

    Science.gov (United States)

    Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

    2011-12-01

    Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

    Science.gov (United States)

    Gong, Jin-Song; Li, Wei; Zhang, Dan-Dan; Xie, Min-Feng; Yang, Biao; Zhang, Rong-Xian; Li, Heng; Lu, Zhen-Ming; Xu, Zheng-Hong; Shi, Jin-Song

    2015-12-17

    In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

  16. Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis

    Directory of Open Access Journals (Sweden)

    Jin-Song Gong

    2015-12-01

    Full Text Available In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-l-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba2+. This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

  17. HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene.

    Science.gov (United States)

    Grably, Melanie R; Stanhill, Ariel; Tell, Osnat; Engelberg, David

    2002-04-01

    In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5' deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Deltamsn4Delta cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Delta cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Deltamsn2Deltamsn4Delta cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

  18. Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A.

    Science.gov (United States)

    Stornetta, Alessia; Deng, Kai-Cheng Kieren; Danielli, Sara; Liyanage, H D Sarath; Sturla, Shana J; Wilson, William R; Gu, Yongchuan

    2018-04-07

    PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1 h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (γH2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R 2  = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

  20. Effect of detergents, trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D.

    Science.gov (United States)

    Madyarov, Sh R

    2014-07-01

    The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca2+>Sr2+>Ba2+>Mg2+. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me2+-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.

  1. Apoptotic effect of cordycepin combined with cisplatin and/or paclitaxel on MA-10 mouse Leydig tumor cells

    Directory of Open Access Journals (Sweden)

    Kang FC

    2015-09-01

    Full Text Available Fu-Chi Kang,1 Pei-Jung Chen,2 Bo-Syong Pan,2,3 Meng-Shao Lai,2,3 Yung-Chia Chen,4 Bu-Miin Huang2,31Department of Anesthesia, Chi Mei Medical Center, Chiali, 2Department of Cell Biology and Anatomy, 3Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, 4Department of Anatomy, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China Background: Chemotherapy is not limited to a single treatment, and the evidence demonstrates that different drug combinations can have positive results in patients. In this study, we sought to determine whether cordycepin combined with cisplatin and/or paclitaxel would have an additive effective on inducing apoptosis in mouse Leydig tumor cells, and the mechanisms were also briefly examined.Methods: The additive effects of cordycepin combined with cisplatin and/or paclitaxel on apoptosis in MA-10 cells were investigated by monitoring changes in morphological characteristics and examining cell viability, flow cytometry assays, and Western blot analyses.Results: Combination of cordycepin plus cisplatin and/or paclitaxel for 12 and 24 hours induced apoptotic features in MA-10 cells. The MTT assay showed that the combination treatment reduced the viability of MA-10 cells in a dose-dependent manner, with additive effects. Cell cycle analysis showed that combination treatment significantly increased subG1 phase cell numbers in MA-10 cells, indicating apoptosis. Moreover, cordycepin plus cisplatin and/or paclitaxel significantly induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase, and phosphorylation of c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, p38, and p53 proteins in MA-10 cells.Conclusion: Cordycepin plus cisplatin and/or paclitaxel can have an additive effect on apoptosis in MA-10 cells, with activation of caspase, mitogen-activated protein kinase, and p53 signal pathways. Keywords: cordycepin

  2. Silica-supported Macroporous Chitosan Bead for Affinity Purification of Trypsin Inhibitor

    Institute of Scientific and Technical Information of China (English)

    Feng Na XI; Jian Min WU; Ming Ming LUAN

    2005-01-01

    Macroporous cross-linking chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG) molecular imprinting methods. Formation of macroporous surface was investigated by scanning electron microscopy (SEM) and BET analysis.The prepared bead was activated by reacting with 1,2-ethylene diglycidyl ether for introducing epoxy groups, and trypsin could be efficiently immobilized on the bead as a biospecific ligand.The bead bearing trypsin was employed to purify trypsin inhibitor (TIs) from egg white as affinity adsorbent.

  3. A novel synthetic analog of militarin, MA-1 induces mitochondrial dependent apoptosis by ROS generation in human lung cancer cells

    International Nuclear Information System (INIS)

    Yoon, Deok Hyo; Lim, Mi-Hee; Lee, Yu Ran; Sung, Gi-Ho; Lee, Tae-Ho; Jeon, Byeong Hwa; Cho, Jae Youl; Song, Won O.; Park, Haeil; Choi, Sunga; Kim, Tae Woong

    2013-01-01

    A synthetic Militarin analog-1[(2R,3R,4R,5R)-1,6-bis(4-(2,4,4-trimethylpentan-2-yl)phenoxy) hexane-2,3,4,5-tetraol] is a novel derivative of constituents from Cordyceps militaris, which has been used to treat a variety of chronic diseases including inflammation, diabetes, hyperglycemia and cancers. Here, we report for the first time the synthesis of Militarin analog-1 (MA-1) and the apoptotic mechanism of MA-1 against human lung cancer cell lines. Treatment with MA-1 significantly inhibited the viability of 3 human lung cancer cell lines. The inhibition of viability and growth in MA-1-treated A549 cells with an IC 50 of 5 μM were mediated through apoptosis induction, as demonstrated by an increase in DNA fragmentation, sub-G 0 /G 1 -DNA fraction, nuclear condensation, and phosphatidylserine exposure. The apoptotic cell death caused mitochondrial membrane permeabilization through regulation of expression of the Bcl-2 family proteins, leading to cytochrome c release in a time-dependent manner. Subsequently, the final stage of apoptosis, activation of caspase-9/-3 and cleavage of poly (ADP ribose) polymerase, was induced. Furthermore, A549 lung cancer cells were more responsive to MA-1 than a bronchial epithelial cell line (BEAS-2B), involving the rapid generation of reactive oxygen species (ROS), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation. The pharmacological inhibition of ROS generation and JNK/p38 MAPK exhibited attenuated DNA fragmentation in MA-1-induced apoptosis. Oral administration of MA-1 also retarded growth of A549 orthotopic xenografts. In conclusion, the present study indicates that the new synthetic derivative MA-1 triggers mitochondrial apoptosis through ROS generation and regulation of MAPKs and may be a potent therapeutic agent against human lung cancer. - Highlights: • We report a novel synthesized derivative, militarin analog-1 (MA-1). • MA-1-induced cancer cell death was triggered by the ROS

  4. A novel synthetic analog of militarin, MA-1 induces mitochondrial dependent apoptosis by ROS generation in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Deok Hyo; Lim, Mi-Hee [Department of Biochemistry, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Yu Ran [Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Sung, Gi-Ho [Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 404-707 (Korea, Republic of); Lee, Tae-Ho [R and D Center, Dong-A Pharmaceutical Co, Ltd, Yongin 446-905 (Korea, Republic of); Jeon, Byeong Hwa [Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Cho, Jae Youl [Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Song, Won O. [Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States); Park, Haeil [College of Pharmacy, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Choi, Sunga, E-mail: sachoi@cnu.ac.kr [Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Kim, Tae Woong, E-mail: tawkim@kangwon.ac.kr [Department of Biochemistry, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2013-12-15

    A synthetic Militarin analog-1[(2R,3R,4R,5R)-1,6-bis(4-(2,4,4-trimethylpentan-2-yl)phenoxy) hexane-2,3,4,5-tetraol] is a novel derivative of constituents from Cordyceps militaris, which has been used to treat a variety of chronic diseases including inflammation, diabetes, hyperglycemia and cancers. Here, we report for the first time the synthesis of Militarin analog-1 (MA-1) and the apoptotic mechanism of MA-1 against human lung cancer cell lines. Treatment with MA-1 significantly inhibited the viability of 3 human lung cancer cell lines. The inhibition of viability and growth in MA-1-treated A549 cells with an IC{sub 50} of 5 μM were mediated through apoptosis induction, as demonstrated by an increase in DNA fragmentation, sub-G{sub 0}/G{sub 1}-DNA fraction, nuclear condensation, and phosphatidylserine exposure. The apoptotic cell death caused mitochondrial membrane permeabilization through regulation of expression of the Bcl-2 family proteins, leading to cytochrome c release in a time-dependent manner. Subsequently, the final stage of apoptosis, activation of caspase-9/-3 and cleavage of poly (ADP ribose) polymerase, was induced. Furthermore, A549 lung cancer cells were more responsive to MA-1 than a bronchial epithelial cell line (BEAS-2B), involving the rapid generation of reactive oxygen species (ROS), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation. The pharmacological inhibition of ROS generation and JNK/p38 MAPK exhibited attenuated DNA fragmentation in MA-1-induced apoptosis. Oral administration of MA-1 also retarded growth of A549 orthotopic xenografts. In conclusion, the present study indicates that the new synthetic derivative MA-1 triggers mitochondrial apoptosis through ROS generation and regulation of MAPKs and may be a potent therapeutic agent against human lung cancer. - Highlights: • We report a novel synthesized derivative, militarin analog-1 (MA-1). • MA-1-induced cancer cell death was triggered by

  5. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    International Nuclear Information System (INIS)

    Chhabra, Hemlata; Gupta, Priyanka; Verma, Paul J.; Jadhav, Sameer; Bellare, Jayesh R.

    2014-01-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold

  6. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  7. Immobilization of trypsin on sub-micron skeletal polymer monolith

    Energy Technology Data Exchange (ETDEWEB)

    Yao Chunhe [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Qi Li, E-mail: qili@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hu Wenbin [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Wang Fuyi [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Yang Gengliang [College of Pharmacy, Hebei University, Baoding 071002 (China)

    2011-04-29

    A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-{alpha}-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-{alpha}-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30 deg. C, which is comparable to 24 h digestion in solution at 37 {sup o}C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.

  8. Why Ser and not Thr brokers catalysis in the trypsin fold.

    Science.gov (United States)

    Pelc, Leslie A; Chen, Zhiwei; Gohara, David W; Vogt, Austin D; Pozzi, Nicola; Di Cera, Enrico

    2015-02-24

    Although Thr is equally represented as Ser in the human genome and as a nucleophile is as good as Ser, it is never found in the active site of the large family of trypsin-like proteases that utilize the Asp/His/Ser triad. The molecular basis of the preference of Ser over Thr in the trypsin fold was investigated with X-ray structures of the thrombin mutant S195T free and bound to an irreversible active site inhibitor. In the free form, the methyl group of T195 is oriented toward the incoming substrate in a conformation seemingly incompatible with productive binding. In the bound form, the side chain of T195 is reoriented for efficient substrate acylation without causing steric clash within the active site. Rapid kinetics prove that this change is due to selection of an active conformation from a preexisting ensemble of reactive and unreactive rotamers whose relative distribution determines the level of activity of the protease. Consistent with these observations, the S195T substitution is associated with a weak yet finite activity that allows identification of an unanticipated important role for S195 as the end point of allosteric transduction in the trypsin fold. The S195T mutation abrogates the Na(+)-dependent enhancement of catalytic activity in thrombin, activated protein C, and factor Xa and significantly weakens the physiologically important allosteric effects of thrombomodulin on thrombin and of cofactor Va on factor Xa. The evolutionary selection of Ser over Thr in trypsin-like proteases was therefore driven by the need for high catalytic activity and efficient allosteric regulation.

  9. Probiotic Properties and Cellular Antioxidant Activity of Lactobacillus plantarum MA2 Isolated from Tibetan Kefir Grains.

    Science.gov (United States)

    Tang, Wei; Li, Chao; He, Zengguo; Pan, Fen; Pan, Shuo; Wang, Yanping

    2017-11-20

    Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibetan kefir grains. Its antioxidant properties had been demonstrated in vitro and in vivo previously. In the present study, the probiotic characteristics of this strain were further evaluated by investigating its acid and bile salt tolerances, cell surface hydrophobicity, and autoaggregation, respectively. In addition, the cellular antioxidant activity (CAA) assay was applied to test the antioxidant capacity of the isolate in different growth phases. Same method was also used to evaluate the antioxidant capacity of its fermentation supernatant, cell-free extract, and intact cell quantitatively. The results of probiotic characteristic tests showed that MA2 could survive at pH 2.5 and 0.3% bile salt. Meanwhile, the measurements of cell surface hydrophobicity and autoaggregation were 45.29 ± 2.15 and 6.30 ± 0.34%, respectively. The results of cellular antioxidant activity tests indicated that MA2 had high antioxidant potential. The CAA value of logarithmic phase cell-free extract of MA2 (39,450.00 ± 424.05 μmol quercetin equivalents/100 g sample) was significantly higher than that in stationary phase cell-free extract (3395.98 ± 126.06 μmol quercetin equivalents/100 g sample) and that of fermentation supernatant in logarithmic phase (2174.41 ± 224.47 μmol quercetin equivalents/100 g sample) (p < 0.05). The CAA method was successively applied to evaluate the antioxidant capacity of MA2 in this study, which suggests that it could be used as a useful method for lactic acid bacteria antioxidant potential evaluation.

  10. Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.

    Science.gov (United States)

    Franco, Octávio L; Grossi de Sá, Maria F; Sales, Maurício P; Mello, Luciane V; Oliveira, Adeliana S; Rigden, Daniel J

    2002-11-15

    Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases. Copyright 2002 Wiley-Liss, Inc.

  11. Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Iu. А. Gordiienko

    2014-12-01

    Full Text Available Activity of trypsin-like enzymes (ATLE and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP. In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

  12. Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy.

    Science.gov (United States)

    Gordiienko, Iu A; Babets, Ya V; Kulinich, A O; Shevtsova, A I; Ushakova, G O

    2014-01-01

    Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

  13. Role of trypsin in the replication of Avian metapneumovirus subtype C (strain MN-2a) and its entry into the Vero cells.

    Science.gov (United States)

    Paudel, Sarita; Shin, Hyun-Jin

    2015-12-01

    To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p < 0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Radioimmunoassay of trypsin

    International Nuclear Information System (INIS)

    Stagg, B.H.; Wood, T.P.

    1979-01-01

    This review describes the development and application of a novel test to determine levels of human immunoreactive trypsin, an enzyme produced solely by the pancreas, in biological fluids. Being organ-specific, the assay of immunoreactive trypsin should be an ideal marker of pancreatic function, and this is supported by the results of a number of clinical and research investigations. Use of this assay in studies of chronic pancreatitis, juvenile-onset diabetes, and cystic fibrosis, has yielded much valuable data, and it is expected that further research will lead to and improved understanding of these and other conditions associated with the pancreas in health and disease. (author)

  15. Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma.

    Science.gov (United States)

    Abbattista, Maria R; Jamieson, Stephen M F; Gu, Yongchuan; Nickel, Jennifer E; Pullen, Susan M; Patterson, Adam V; Wilson, William R; Guise, Christopher P

    2015-01-01

    PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.

  16. NuMA and nuclear lamins are cleaved during viral infection - inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death

    International Nuclear Information System (INIS)

    Taimen, Pekka; Berghaell, Heidi; Vainionpaeae, Raija; Kallajoki, Markku

    2004-01-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death

  17. Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

    International Nuclear Information System (INIS)

    Lague, Eric; Tremblay, Jacques J.

    2009-01-01

    There are increasing evidence in the literature reporting the detrimental effects of endocrine disruptors on the development and function of the male reproductive system. One example is cryptorchidism, or undescended testis, caused by exposure to excessive estrogens. Estrogens, acting through the estrogen receptor α (ERα), have been shown to repress expression of the gene encoding insulin-like 3 (INSL3), a small peptide produced by testicular Leydig cells that is essential for normal testis descent. The molecular mechanism of estrogen/ER action on Insl3 expression, however, remains poorly understood. Here we report estradiol (E 2 ) represses Insl3 mRNA levels in MA-10 cells, a Leydig cell line model. We also found that E 2 represses the activity of the human and mouse Insl3 promoter in these cells. The E 2 -responsive region of the human INSL3 promoter was located to the proximal INSL3 promoter. This region does not contain a consensus estrogen response element indicating an indirect mechanism of action. In agreement with this, we found that E 2 -responsiveness was lost when two previously characterized binding sites for the nuclear receptors NUR77 and SF1 were mutated. Finally we show that the E 2 repressive effect could be overcome by cotreatment with testosterone, a positive regulator of Insl3 transcription. Collectively our data provide important new insights into the molecular mechanism of estrogen action in Insl3 transcription in Leydig cells

  18. In vitro and in silico investigations of the binding interactions between chlorophenols and trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yan-Qing, E-mail: wyqing76@126.com [Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng City 224002, Jiangsu Province (China); Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Tan, Chun-Yun [Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Zhuang, Shu-Lin [Institute of Environmental Science, College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058 (China); Zhai, Peng-Zhan; Cui, Yun; Zhou, Qiu-Hua; Zhang, Hong-Mei [Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China); Fei, Zhenghao [Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng City 224002, Jiangsu Province (China); Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City 224002, Jiangsu Province (China)

    2014-08-15

    Graphical abstract: - Highlights: • Binding interactions of five chlorophenols with trypsin were investigated. • The number of chlorine atoms of chlorophenols partly affected the binding ability of them to trypsin. • Noncovalent interactions stabilized the trypsin–chlorophenols complexes. • There was the one main binding site of trypsin for chlorophenols. - Abstract: Being the first-degree toxic pollutants, chlorophenols (CP) have potential carcinogenic and mutagenic activity and toxicity. Since there still lacks studies on molecular interactions of chlorophenols with trypsin, one major binding target of many exogenous environmental pollutants, the binding interactions between five chlorophenols, 2-CP, 2,6-DCP, 2,4,6-TCP, 2,4,6-TCP, 2,3,4,6-TCP and PCP and trypsin were characterized by the combination of multispectroscopic techniques and molecular modeling. The chlorophenols bind at the one main site of trypsin and the binding induces the changes of microenvironment and global conformations of trypsin. Different number of chloride atoms significantly affects the binding and the binding constants K{sub A} ranks as K{sub A} (2-CP) < K{sub A} (2,6-DCP) ≈ K{sub A} (2,4,6-TCP) < K{sub A} (2,3,4,6-TCP) < K{sub A} (PCP). These chlorophenols interacts with trypsin mainly through hydrophobic interactions and via hydrogen bonding interactions and aromatic–aromatic π–π stacking interaction. Our results offer insights into the binding mechanism of chlorophenols with trypsin and provide important information for possible toxicity risk of chlorophenols to human health.

  19. 24 CFR 1000.104 - What families are eligible for affordable housing activities?

    Science.gov (United States)

    2010-04-01

    ... affordable housing activities? 1000.104 Section 1000.104 Housing and Urban Development Regulations Relating... Activities § 1000.104 What families are eligible for affordable housing activities? The following families... Indian area. (b) A non-low income Indian family may receive housing assistance in accordance with § 1000...

  20. Efficiency of inactivation of trypsin inhibitory activity in some selected ...

    African Journals Online (AJOL)

    Trypsin inhibitor (TI) levels in the crop seeds varied between 0.0 in Adansonia digitata and 40.8 TIU/mg in Pterocarpus osun. Efficiency of inactivation of TI by autoclaving ranged from 58.1% in Millettia thonningii to 100% in Sesbania pachycarpa and Lonchocarpus. sericeus. It is concluded that the effect of heat treatment on ...

  1. High-gradient magnetic affinity separation of trypsin from porcine pancreatin

    DEFF Research Database (Denmark)

    Hubbuch, Jürgen; Thomas, Owen R. T.

    2002-01-01

    We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined......-scale studies approximate to95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximate to4.1 at the expense of only a 4% loss in a-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly...

  2. Fish trypsins: potential applications in biomedicine and prospects for production.

    Science.gov (United States)

    Jesús-de la Cruz, Kristal; Álvarez-González, Carlos Alfonso; Peña, Emyr; Morales-Contreras, José Antonio; Ávila-Fernández, Ángela

    2018-04-01

    In fishes, trypsins are adapted to different environmental conditions, and the biochemical and kinetic properties of a broad variety of native isoforms have been studied. Proteolytic enzymes remain in high demand in the detergent, food, and feed industries; however, our analysis of the literature showed that, in the last decade, some fish trypsins have been studied for the synthesis of industrial peptides and for specific biomedical uses as antipathogenic agents against viruses and bacteria, which have been recently patented. In addition, innovative strategies of trypsin administration have been studied to ensure that trypsins retain their properties until they exert their action. Biomedical uses require the production of high-quality enzymes. In this context, the production of recombinant trypsins is an alternative. For this purpose, E. coli -based systems have been tested for the production of fish trypsins; however, P. pastoris -based systems also seem to show great potential in the production of fish trypsins with higher production quality. On the other hand, there is a lack of information regarding the specific structures, biochemical and kinetic properties, and characteristics of trypsins produced using heterologous systems. This review describes the potential uses of fish trypsins in biomedicine and the enzymatic and structural properties of native and recombinant fish trypsins obtained to date, outlining some prospects for their study.

  3. Rapid and Efficient Protein Digestion using Trypsin Coated Magnetic Nanoparticles under Pressure Cycles

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byoungsoo; Lopez-Ferrer, Daniel; Kim, Byoung Chan; Na, Hyon Bin; Park, Yong Il; Weitz, Karl K.; Warner, Marvin G.; Hyeon, Taeghwan; Lee, Sang-Won; Smith, Richard D.; Kim, Jungbae

    2011-01-01

    Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple crosslinking of the enzyme to magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while a control sample of covalently-attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. Digestions were carried out on a single model protein, a five protein mixture, and a whole mouse brain proteome, and also compared for digestion at atmospheric pressure and 37 ºC for 12 h, and in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, the EC-TR/NPs performed equally as well or better than free trypsin in terms of the number of peptide/protein identifications and reproducibility across technical replicates. However, the concomitant use of EC-TR/NPs and PCT resulted in very fast (~1 min) and more reproducible digestions.

  4. Chronostratigraphy of KNM-ER 3733 and other Area 104 hominins from Koobi Fora.

    Science.gov (United States)

    Lepre, Christopher J; Kent, Dennis V

    2015-09-01

    A magnetostratigraphy for ∼ 60 m of Koobi Fora Formation sediment in Area 104 was derived from 46 oriented samples that produced well-resolved characteristic magnetizations from progressive thermal demagnetization. Approximately 59 m below the Morte Tuff, previously dated to ∼ 1.51 Ma (millions of years ago), the Olduvai-Matuyama boundary (∼ 1.78 Ma) was found to be at the level of marker bed A2--inconsistent with the Area 102 type section and thus contrary to fossil dating schemes that utilize temporal equivalence between A2 [104] and A2 [102]. The magnetostratigraphic data, coupled with the Morte Tuff, provide a means to interpolate new ages for marker beds A2 [104] and the White Tuff, as well as multiple Area 104 hominin fossils. Noteworthy is the new date of ∼ 1.63 Ma for KNM-ER 3733, which now implicates KNM-ER 2598 as the sole early African Homo erectus fossil demonstrably older than Dmanisi and Java Homo specimens. Re-dating KNM-ER 3733 creates a ∼ 300-kyr gap at 1.9 to 1.6 Ma in the African fossil record of H. erectus, which might be partially spanned by hand axes recently dated at ∼ 1.76 Ma, if the Acheulian is indeed proprietary to this species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Maciel, J. C., E-mail: jackeline-maciel@hotmail.com [Universidade Federal de Roraima (Brazil); Mercês, A. A. D.; Cabrera, M. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil); Shigeyosi, W. T. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Souza, S. D. de; Olzon-Dionysio, M.; Fabris, J. D. [Universidade Federal dos Vales de Jequitinhonha e Mucuri (Brazil); Cardoso, C. A. [Universidade Federal de São Carlos, Departamento de Física (Brazil); Neri, D. F. M. [Universidade Federal do Vale do São Francisco (Brazil); Silva, M. P. C.; Carvalho, L. B. [Universidade Federal de Pernambuco, Laboratório de Imunopatologia Keizo Asami (Brazil)

    2016-12-15

    It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe {sup 3+} ions and their preparation by coating them with polyaniline. The electronic micrograph analysis showed that the mean diameter for the nanoparticles is ∼15 nm. FTIR, powder X-ray diffraction and Mössbauer spectroscopy were used to understand the chemical, crystallographic and {sup 57}Fe hyperfine structures for the two samples. The nanoparticles, which exhibited magnetic behavior with relatively high spontaneous magnetization at room temperature, were identified as being mainly formed by maghemite (γFe{sub 2}O{sub 3}). The coated magnetic nanoparticles (sample labeled “mPANI”) presented a real ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative (sample “mPANIG-Trypsin”). The amount of protein and specific activity of the immobilized trypsin were found to be 13±5 μg of protein/mg of mPANI (49.3 % of immobilized protein) and 24.1±0.7 U/mg of immobilized protein, respectively. After 48 days of storage at 4 {sup ∘}C, the activity of the immobilized trypsin was found to be 89 % of its initial activity. This simple, fast and low-cost procedure was revealed to be a promising way to prepare mPANI nanoparticles if technological applications addressed to covalently link biomolecules are envisaged. This route yields chemically stable derivatives, which can be easily recovered from the reaction mixture with a magnetic field and recyclable reused.

  6. Enzymatic synthesis of gold nanoflowers with trypsin

    International Nuclear Information System (INIS)

    Li Linmei; Weng Jian

    2010-01-01

    A one-step and eco-friendly approach for the room-temperature synthesis of trypsin-mediated three-dimensional (3D) gold nanoflowers (AuNFs) with high colloidal stability is demonstrated. To prepare AuNFs, ascorbic acid (AA) was quickly added into the premixed solution of HAuCl 4 and trypsin at pH = 5.0. The results show that the molar ratio and feeding order of reactant agents, pH and reaction time play important roles in the formation of NFs. The growth mechanism of AuNFs is suggested as three steps: (1) immobilization of AuCl 4 - ions with a positively charged trypsin template, (2) spontaneous reduction of AuCl 4 - ions with AA in situ and capping Au 0 by 12 cysteines of trypsin, (3) reduction of more AuCl 4 - ions on the Au nuclei formed in the initial stages and anisotropic growth into AuNFs.

  7. Dioscorin, the major tuber storage protein of yam (Dioscorea batatas decne) with carbonic anhydrase and trypsin inhibitor activities.

    Science.gov (United States)

    Hou, W C; Liu, J S; Chen, H J; Chen, T E; Chang, C F; Lin, Y H

    1999-05-01

    Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

  8. A novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin

    International Nuclear Information System (INIS)

    Xu, Kaijia; Wang, Yuzhi; Li, Yixue; Lin, Yunxuan; Zhang, Haibao; Zhou, Yigang

    2016-01-01

    Novel poly(deep eutectic solvent) grafted silica-coated magnetic microspheres (Fe 3 O 4 @SiO 2 -MPS@PDES) were prepared by polymerization of choline chloride-itaconic acid (ChCl-IA) and γ-MPS-modified magnetic silica composites, and were characterized by vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectra (XPS), thermal gravimetric analysis (TGA) and transmission electron microscope (TEM). Then the synthetic Fe 3 O 4 @SiO 2 -MPS@PDES microspheres were applied for the magnetic solid-phase extraction (MSPE) of trypsin for the first time. After extraction, the concentration of trypsin in the supernatant was determined by a UV–vis spectrophotometer. Single factor experiments were carried out to investigate the effects of the extraction process, including the concentration of trypsin, the ionic strength, the pH value, the extraction time and the temperature. Experimental results showed the extraction capacity could reach up to 287.5 mg/g under optimized conditions. In comparison with Fe 3 O 4 @SiO 2 -MPS, Fe 3 O 4 @SiO 2 -MPS@PDES displayed higher extraction capacity and selectivity for trypsin. According to the regeneration studies, Fe 3 O 4 @SiO 2 -MPS@PDES microspheres can be recycled six times without significant loss of its extraction capacity, and retained a high extraction capacity of 233 mg/g after eight cycles. Besides, the activity studies also demonstrated that the activity of the extracted trypsin was well retained. Furthermore, the analysis of real sample revealed that the prepared magnetic microspheres can be used to purify trypsin in crude bovine pancreas extract. These results highlight the potential of the proposed Fe 3 O 4 @SiO 2 -MPS@PDES-MSPE method in separation of biomolecules. - Highlights: • A strategy for solid-phase extraction of trypsin based on poly(deep eutectic solvent) modified magnetic silica microspheres. • Fe 3 O 4 @SiO 2 -MPS@PDES showed higher extraction capacity

  9. In Vitro Antiviral Activity of Rubia Cordifolia Aerial Part Extract Against Rotavirus

    Directory of Open Access Journals (Sweden)

    Yuanyuan Sun

    2016-09-01

    Full Text Available The root of Rubia cordifolia (R. cordifolia has been used traditionally as a hemostatic agent, while the aerial part of the plant consisting of leaf and stem is known to exhibit anti-diarrheal properties and has been widely used as a remedy in many parts of China. As rotavirus is one of the most commonly associated diarrhea-causing pathogen, this study aims to investigate the anti-rotaviral effect of R. cordifolia aerial part (RCAP. The cytotoxicity of RCAP towards MA-104 cells was evaluated using the WST-8 assay. Colloidal gold method and real time polymerase chain reaction (qPCR assay were used to confirm the findings of the antiviral assay. Then, 4',6-diamidino-2-phenylindole (DAPI staining method was subsequently used to investigate the mode of death among the cells. And the representative components of aqueous extract were isolated and identified. It was shown that both the viability of MA-104 cells and the viral load were reduced with increasing concentration of the extract. DAPI staining showed that virus-induced apoptosis was the cause of the low cell viability and viral load, an effect which was accelerated with incubation in the aqueous herbal extract. The major compounds postulated to exhibit this activity were isolated from the aqueous herbal extract and identified to be compounds Xanthopurpurin and Vanillic Acid. This study showed that RCAP extract effectively inhibited rotavirus multiplication by promoting virus-induced apoptosis in MA-104 cells.

  10. Gamma rays induced mutation for low phytic acid and trypsin inhibitor content in soybean

    International Nuclear Information System (INIS)

    Gupta, S.K.; Manjaya, J.G.

    2017-01-01

    Soybean (Glycine max (L.) Merrill) is an important source of vegetable protein and is used as a food, feed and health supplement. However, consumption of soybean as food is limited because of the presence of many anti-nutritional factors. Trypsin inhibitors and phytic acid are two major anti-nutritional factors present in soybean that need to be removed for increasing the soybean consumption as food. Trypsin inhibitor is known to inhibit the trypsin/chymotrpsin activity and phytic acid reduces the bioavailability of essential micronutrients in digestive tract, resulting in adverse effect on health. Therefore, developing soybean cultivars having low trypsin inhibitors and phytic acid content is highly desirable. Soybean cultivar JS 93-05 was irradiated with 250 Gy gamma rays to induce mutation for various morphological and biochemical characters. A large number of mutants with altered morphological characters were identified. Ninety true breeding mutant lines in M6 generation were screened for trypsin inhibitor and phytic acid content. The phytic acid content was estimated using modified colorimetric method and trypsin inhibitor concentration was estimated using BAPNA as substrate in colorimetric method. The phytic acid content in the mutants varied from 7.59 to 24.14 mg g -1 . Two mutants lines TSG - 62 (7.59 mg g -1 ) and TSG - 66 (9.62 mg g -1 ) showed significant low phytic acid content as compared to the parent JS 93-05 (20.19 mg g -1 ). The trypsin inhibitor concentration in the mutants varied from 19.92 to 53.64 TIU mg -1 and one mutant line (TSG -14) was found with the lowest trypsin inhibitor concentration of 19.92 TIU mg -1 compared to parent JS 93-05 (50.90 TIU mg -1 ). The mutant lines identified in this study will serve as important genetic resources for developing low phytic acid and low trypsin inhibitor cultivars in soybean. (author)

  11. Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization.

    Science.gov (United States)

    Villalba-Villalba, Ana Gloria; Ramírez-Suárez, Juan Carlos; Valenzuela-Soto, Elisa Miriam; Sánchez, Guillermina García; Ruiz, Gisela Carvallo; Pacheco-Aguilar, Ramón

    2013-11-15

    Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  13. Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.

    Science.gov (United States)

    Zhao, Mingzhi; Wu, Feilin; Xu, Ping

    2015-12-01

    Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Difference in TB10.4 T-cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Billeskov, Rolf; Grandal, Michael V; Poulsen, Christian

    2010-01-01

    vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti-tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4(+) T-cell specific TB10.4 epitope-pattern, which differed completely from...... that induced by recombinant TB10.4. This difference was not due to post-translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed...... that both TB10.4 and BCG were transported to Lamp(+)-compartments. BCG and TB10.4 however, were directed to different types of Lamp(+)-compartments in the same APC, which may lead to different epitope recognition patterns. In conclusion, we show that different vectors can induce completely different...

  15. Adiponectin influences progesterone production from MA-10 Leydig cells in a dose-dependent manner.

    Science.gov (United States)

    Landry, David; Paré, Aurélie; Jean, Stéphanie; Martin, Luc J

    2015-04-01

    Obesity in men is associated with lower testosterone levels, related to reduced sperm concentration and the development of various diseases with aging. Hormones produced by the adipose tissue may have influences on both metabolism and reproductive function. Among them, the production and secretion of adiponectin is inversely correlated to total body fat. Adiponectin receptors (AdipoR1 and AdipoR2) have been found to be expressed in testicular Leydig cells (producing testosterone). Since StAR and Cyp11a1 are essential for testosterone synthesis and adiponectin has been shown to regulate StAR mRNA in swine granulosa cells, we hypothesized that adiponectin might also regulate these genes in Leydig cells. Our objective was to determine whether adiponectin regulates StAR and Cyp11a1 genes in Leydig cells and to better define its mechanisms of action. Methods used in the current study are qPCR for the mRNA levels, transfections for promoter activities, and enzyme-linked immunosorbent assay for the progesterone concentration. We have found that adiponectin cooperates with cAMP-dependent stimulation to activate StAR and Cyp11a1 mRNA expressions in a dose-dependent manner in MA-10 Leydig cells as demonstrated by transfection of a luciferase reporter plasmid. These results led to a significant increase in progesterone production from MA-10 cells. Thus, our data suggest that high doses of adiponectin typical of normal body weight may promote testosterone production from Leydig cells.

  16. RITA plus 3-MA overcomes chemoresistance of head and neck cancer cells via dual inhibition of autophagy and antioxidant systems.

    Science.gov (United States)

    Shin, Daiha; Kim, Eun Hye; Lee, Jaewang; Roh, Jong-Lyel

    2017-10-01

    Reactivation of p53 and induction of tumor cell apoptosis (RITA) is a small molecule that blocks p53-MDM2 interaction, thereby reactivating p53 in tumors. RITA can induce exclusive apoptosis in cancer cells independently of the p53 pathway; however, the resistance of cancer cells remains a major drawback. Here, we found a novel resistance mechanism of RITA treatment and an effective combined treatment to overcome RITA resistance in head and neck cancer (HNC) cells. The effects of RITA and 3-methyladenine (3-MA) were tested in different HNC cell lines, including cisplatin-resistant and acquired RITA-resistant HNC cells. The effects of each drug alone and in combination were assessed by measuring cell viability, apoptosis, cell cycle, glutathione, reactive oxygen species, protein expression, genetic inhibition of p62 and Nrf2, and a mouse xenograft model of cisplatin-resistant HNC. RITA induced apoptosis of HNC cells at different levels without significantly inhibiting normal cell viability. Following RITA treatment, RITA-resistant HNC cells exhibited a sustained expression of other autophagy-related proteins, overexpressed p62, and displayed activation of the Keap1-Nrf2 antioxidant pathway. The autophagy inhibitor 3-MA sensitized resistant HNC cells to RITA treatment via the dual inhibition of molecules related to the autophagy and antioxidant systems. Silencing of the p62 gene augmented the combined effects. The effective antitumor activity of RITA plus 3-MA was also confirmed in vivo in mouse xenograft models transplanted with resistant HNC cells, showing increased oxidative stress and DNA damage. The results indicate that RITA plus 3-MA can help overcome RITA resistance in HNC cells. This study revealed a novel RITA resistant mechanism associated with the sustained induction of autophagy, p62 overexpression, and Keap1-Nrf2 antioxidant system activation. The combined treatment of RITA with the autophagy inhibitor 3-methyladenine overcomes RITA resistance via dual

  17. Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III).

    Science.gov (United States)

    Lesner, A; Brzozowski, K; Kupryszewski, G; Rolka, K

    2000-03-05

    A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme. Copyright 2000 Academic Press.

  18. A novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Kaijia [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Li, Yixue; Lin, Yunxuan; Zhang, Haibao [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082 (China); Zhou, Yigang [Department of Microbiology, College of Basic Medicine, Central South University, Changsha, 410083 (China)

    2016-11-23

    Novel poly(deep eutectic solvent) grafted silica-coated magnetic microspheres (Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS@PDES) were prepared by polymerization of choline chloride-itaconic acid (ChCl-IA) and γ-MPS-modified magnetic silica composites, and were characterized by vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectra (XPS), thermal gravimetric analysis (TGA) and transmission electron microscope (TEM). Then the synthetic Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS@PDES microspheres were applied for the magnetic solid-phase extraction (MSPE) of trypsin for the first time. After extraction, the concentration of trypsin in the supernatant was determined by a UV–vis spectrophotometer. Single factor experiments were carried out to investigate the effects of the extraction process, including the concentration of trypsin, the ionic strength, the pH value, the extraction time and the temperature. Experimental results showed the extraction capacity could reach up to 287.5 mg/g under optimized conditions. In comparison with Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS, Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS@PDES displayed higher extraction capacity and selectivity for trypsin. According to the regeneration studies, Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS@PDES microspheres can be recycled six times without significant loss of its extraction capacity, and retained a high extraction capacity of 233 mg/g after eight cycles. Besides, the activity studies also demonstrated that the activity of the extracted trypsin was well retained. Furthermore, the analysis of real sample revealed that the prepared magnetic microspheres can be used to purify trypsin in crude bovine pancreas extract. These results highlight the potential of the proposed Fe{sub 3}O{sub 4}@SiO{sub 2}-MPS@PDES-MSPE method in separation of biomolecules. - Highlights: • A strategy for solid-phase extraction of trypsin based on poly(deep eutectic solvent) modified magnetic silica

  19. Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

    Science.gov (United States)

    Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

    2014-10-01

    We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for

  20. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    Directory of Open Access Journals (Sweden)

    Côté Claude H

    2011-10-01

    Full Text Available Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2. The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs. Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2, a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream

  1. Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity.

    Science.gov (United States)

    Pimenta de Oliveira, Sofia; Dantas de Oliveira, Caroline; Viana Sant'Anna, Mauricio Roberto; Carneiro Dutra, Heverton Leandro; Caragata, Eric Pearce; Moreira, Luciano Andrade

    2017-08-01

    Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Low molecular weight squash trypsin inhibitors from Sechium edule seeds.

    Science.gov (United States)

    Laure, Hélen J; Faça, Vítor M; Izumi, Clarice; Padovan, Júlio C; Greene, Lewis J

    2006-02-01

    Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.

  3. Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization

    Energy Technology Data Exchange (ETDEWEB)

    Giraldo-Gomez, D.M., E-mail: davidmauro2008@gmail.com [Posgrado en Ciencia e Ingeniería de Materiales, Universidad Nacional Autónoma de México (UNAM), Unidad de Posgrado Edificio “C” 1er Piso, Circuito de Posgrados, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D. F., México (Mexico); Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); Leon-Mancilla, B. [Departamento de Cirugía, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Edificio “D” Planta Baja, Circuito Interior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); Del Prado-Audelo, M.L. [Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); and others

    2016-02-01

    There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15 consecutive cycles were performed over 32 days. Protocol B (a variation of Protocol A) — EDTA being added — with 15 consecutive cycles performed over 60 days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5 consecutive cycles being performed over 10 days. For the three protocols, hematoxylin–eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix

  4. Regulation of the Hsp104 middle domain activity is critical for yeast prion propagation.

    Directory of Open Access Journals (Sweden)

    Jennifer E Dulle

    Full Text Available Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria. We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.

  5. Chemical repair of trypsin-histidinyl radical

    International Nuclear Information System (INIS)

    Jovanovic, S.V.; Ruvarac, I.; Jankovic, I.; Josimovic, L.

    1991-01-01

    Oxyl radicals, such as hydroxyl, alkoxyl and peroxyl, react with biomolecules to produce bioradicals. Unless chemically repaired by suitable antioxidants, these bioradicals form stable products. This leads to loss of biological function of parent biomolecules with deleterious biological results, such as mutagenesis and cancer. Consequently, the understanding of the mechanisms of oxyl radical damage to biomolecules and chemical repair of such damage is crucial for the development of strategies for anticarcinogenesis and radioprotection. In this study the chemical repair of the histidinyl radical generated upon the trichloromethylperoxyl radical reaction with trypsin vas investigated by gamma radiolysis. The trypsin histidinyl radical is a resonance-stabilized heterocyclic free radical which was found to be unreactive with oxygen. The efficacy of the chemical repair of the trypsin-histidinyl radical by endogenous antioxidants which are electron donors (e.g. 5-hydroxytryptophan, uric acid) is compared to that of antioxidants which are H-atom donors (e. g. glutathione). 9 refs., 2 figs., 1 tab

  6. Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation.

    Directory of Open Access Journals (Sweden)

    Yang-Nim Park

    Full Text Available [PSI(+] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+] and inactivation of Hsp104 cures [PSI(+] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.

  7. Anti-Ma2 antibody related paraneoplastic limbic/brain stem encephalitis associated with breast cancer expressing Ma1, Ma2, and Ma3 mRNAs.

    Science.gov (United States)

    Sahashi, K; Sakai, K; Mano, K; Hirose, G

    2003-09-01

    A 69 year old woman presented with cognitive impairment and supranuclear gaze palsy caused by paraneoplastic limbic/brain stem encephalitis associated with atypical medullary breast carcinoma. The cerebrospinal fluid from the patient harboured an anti-neuronal cell antibody against Ma2 antigen, but not against Ma1 or Ma3 antigen. Despite the antibody being restricted to the Ma2 antigen, the patient's cancer tissue expressed Ma1, Ma2, and Ma3 mRNAs. These results, and the expression of Ma2 mRNA in an atypical medullar breast carcinoma in another patient without paraneoplastic encephalitis, indicate that the induction of anti-Ma2 antibody depends on host immunoreponsiveness and not on the presence of the antigen itself in the cancer.

  8. Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

    Science.gov (United States)

    Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

    1995-02-27

    Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

  9. FRET-based modified graphene quantum dots for direct trypsin quantification in urine

    Energy Technology Data Exchange (ETDEWEB)

    Poon, Chung-Yan; Li, Qinghua [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Zhang, Jiali; Li, Zhongping [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan [Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Lee, Albert Wai-Ming; Chan, Wing-Hong [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Li, Hung-Wing, E-mail: hwli@hkbu.edu.hk [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong)

    2016-04-21

    A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening. - Highlights: • A FRET-based biosensor was developed for direct quantification of trypsin. • Fast and sensitive screening of pancreatic disease was facilitated. • The direct quantification of trypsin in urine samples was demonstrated.

  10. In vitro assessment of phthalate acid esters-trypsin complex formation.

    Science.gov (United States)

    Chi, Zhenxing; Zhao, Jing; Li, Weiguo; Araghi, Arash; Tan, Songwen

    2017-10-01

    In this work, interactions of three phthalate acid esters (PAEs), including dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP), with trypsin have been studied in vitro, under simulated physiological conditions using multi-spectroscopic techniques and molecular modeling. The results show that these PAEs can bind to the trypsin, forming trypsin-PAEs complexes, mainly via hydrophobic interactions, with the affinity order of DMP > DEP > DBP. Binding to the PAEs is found to result in molecular deformation of trypsin. The modeling results suggest that only DBP can bind with the amino acid residues of the catalytic triad and S1 binding pocket of trypsin, leading to potential competitive enzyme inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    M.E. Pereira

    2005-11-01

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

  12. Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

    Science.gov (United States)

    Yun, Bingling; Guan, Xiaolu; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2015-10-26

    Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV.

  13. The immobilisation of trypsin and glucose oxidase onto natural rubber-g.co-HEMA - high energy radiation derived copolymeric support systems

    International Nuclear Information System (INIS)

    Devi, S.; Guthrie, J.T.; Beddows, C.G.

    1990-01-01

    Natural rubber has been grafted with 2-HEMA by three different methods each involving Co(60)γ-radiation as the initiation source. The grafted samples were used in the immobilisation of glucose oxidase and trypsin. Optimisation of immobilisation involving trypsin was studied with regard to the pH and the type of crosslinking agent. It was observed that the immobilised enzyme had superior stability over a wider pH range when compared to the free trypsin. The retention of activity demonstrated by the immobilised trypsin was significant. That of immobilised glucose oxidase was far from being satisfactory. (author)

  14. Comparative study of the binding of trypsin to caffeine and theophylline by spectrofluorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruiyong, E-mail: wangry@zzu.edu.cn [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Kang, Xiaohui [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Wang, Ruiqiang [The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 (China); Wang, Rui; Dou, Huanjing; Wu, Jing; Song, Chuanjun [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China); Chang, Junbiao, E-mail: changjunbiao@zzu.edu.cn [Department of Chemistry, Zhengzhou University, Zhengzhou 450001 (China)

    2013-06-15

    The interactions between trypsin and caffeine/theophylline were investigated by fluorescence spectroscopy, UV–visible absorption spectroscopy, resonance light scattering and synchronous fluorescence spectroscopy under mimic physiological conditions. The results revealed that the fluorescence quenching of trypsin by caffeine and theophylline was the result of the formed complex of caffeine–trypsin and theophylline–trypsin. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that caffeine was the stronger quencher and bound to trypsin with higher affinity than theophylline. -- Highlights: ► The fluorescence of trypsin can be quenched by caffeine or theophylline via hydrophobic contacts. ► Caffeine binds to trypsin with higher affinity than theophylline. ► The influence of molecular structure on the binding aspects is reported.

  15. Comparative study of the binding of trypsin to caffeine and theophylline by spectrofluorimetry

    International Nuclear Information System (INIS)

    Wang, Ruiyong; Kang, Xiaohui; Wang, Ruiqiang; Wang, Rui; Dou, Huanjing; Wu, Jing; Song, Chuanjun; Chang, Junbiao

    2013-01-01

    The interactions between trypsin and caffeine/theophylline were investigated by fluorescence spectroscopy, UV–visible absorption spectroscopy, resonance light scattering and synchronous fluorescence spectroscopy under mimic physiological conditions. The results revealed that the fluorescence quenching of trypsin by caffeine and theophylline was the result of the formed complex of caffeine–trypsin and theophylline–trypsin. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that caffeine was the stronger quencher and bound to trypsin with higher affinity than theophylline. -- Highlights: ► The fluorescence of trypsin can be quenched by caffeine or theophylline via hydrophobic contacts. ► Caffeine binds to trypsin with higher affinity than theophylline. ► The influence of molecular structure on the binding aspects is reported

  16. Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots.

    Science.gov (United States)

    Cai, Daguang; Thurau, Tim; Tian, Yanyan; Lange, Tina; Yeh, Kai-Wun; Jung, Christian

    2003-04-01

    Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.

  17. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Liquid crystalline trypsin, Peru balsam, castor... NEW ANIMAL DRUGS § 524.2620 Liquid crystalline trypsin, Peru balsam, castor oil. (a)(1) Specifications... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam...

  18. Disruption of the novel gene fad104 causes rapid postnatal death and attenuation of cell proliferation, adhesion, spreading and migration

    International Nuclear Information System (INIS)

    Nishizuka, Makoto; Kishimoto, Keishi; Kato, Ayumi; Ikawa, Masahito; Okabe, Masaru; Sato, Ryuichiro; Niida, Hiroyuki; Nakanishi, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-01-01

    The molecular mechanisms at the beginning of adipogenesis remain unknown. Previously, we identified a novel gene, fad104 (factor for adipocyte differentiation 104), transiently expressed at the early stage of adipocyte differentiation. Since the knockdown of the expression of fad104 dramatically repressed adipogenesis, it is clear that fad104 plays important roles in adipocyte differentiation. However, the physiological roles of fad104 are still unknown. In this study, we generated fad104-deficient mice by gene targeting. Although the mice were born in the expected Mendelian ratios, all died within 1 day of birth, suggesting fad104 to be crucial for survival after birth. Furthermore, analyses of mouse embryonic fibroblasts (MEFs) prepared from fad104-deficient mice provided new insights into the functions of fad104. Disruption of fad104 inhibited adipocyte differentiation and cell proliferation. In addition, cell adhesion and wound healing assays using fad104-deficient MEFs revealed that loss of fad104 expression caused a reduction in stress fiber formation, and notably delayed cell adhesion, spreading and migration. These results indicate that fad104 is essential for the survival of newborns just after birth and important for cell proliferation, adhesion, spreading and migration

  19. A trypsin inhibitor from Tecoma stans leaves inhibits growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei

    Directory of Open Access Journals (Sweden)

    Leydianne Leite de Siqueira Patriota

    2016-04-01

    Full Text Available Tecoma stans (yellow elder has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI, which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs was evaluated. TesTI was isolated from saline extract by ammonium sulphate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC and fungicide (MFC concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, Ki 43 nM inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 µg/mL. The MFCs were 200 µg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells.

  20. Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with (/sup 3/H)diisopropyl fluorophosphate

    Energy Technology Data Exchange (ETDEWEB)

    Small, D.H.; Chubb, I.W.

    1988-07-01

    In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor (/sup 3/H)diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by (/sup 3/H)diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.

  1. Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

    Science.gov (United States)

    Franco, Octávio L; Dias, Simoni C; Magalhães, Claudio P; Monteiro, Ana C S; Bloch, Carlos; Melo, Francislete R; Oliveira-Neto, Osmundo B; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima

    2004-01-01

    The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.

  2. A novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin.

    Science.gov (United States)

    Xu, Kaijia; Wang, Yuzhi; Li, Yixue; Lin, Yunxuan; Zhang, Haibao; Zhou, Yigang

    2016-11-23

    Novel poly(deep eutectic solvent) grafted silica-coated magnetic microspheres (Fe 3 O 4 @SiO 2 -MPS@PDES) were prepared by polymerization of choline chloride-itaconic acid (ChCl-IA) and γ-MPS-modified magnetic silica composites, and were characterized by vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectra (XPS), thermal gravimetric analysis (TGA) and transmission electron microscope (TEM). Then the synthetic Fe 3 O 4 @SiO 2 -MPS@PDES microspheres were applied for the magnetic solid-phase extraction (MSPE) of trypsin for the first time. After extraction, the concentration of trypsin in the supernatant was determined by a UV-vis spectrophotometer. Single factor experiments were carried out to investigate the effects of the extraction process, including the concentration of trypsin, the ionic strength, the pH value, the extraction time and the temperature. Experimental results showed the extraction capacity could reach up to 287.5 mg/g under optimized conditions. In comparison with Fe 3 O 4 @SiO 2 -MPS, Fe 3 O 4 @SiO 2 -MPS@PDES displayed higher extraction capacity and selectivity for trypsin. According to the regeneration studies, Fe 3 O 4 @SiO 2 -MPS@PDES microspheres can be recycled six times without significant loss of its extraction capacity, and retained a high extraction capacity of 233 mg/g after eight cycles. Besides, the activity studies also demonstrated that the activity of the extracted trypsin was well retained. Furthermore, the analysis of real sample revealed that the prepared magnetic microspheres can be used to purify trypsin in crude bovine pancreas extract. These results highlight the potential of the proposed Fe 3 O 4 @SiO 2 -MPS@PDES-MSPE method in separation of biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Crystallization and preliminary X-ray diffraction studies of Murraya koenigii trypsin inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Shee, Chandan [Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667 (India); Singh, Tej P. [Department of Biophysics, All India Institute of Medical Sciences, New Delhi 100 029 (India); Kumar, Pravindra, E-mail: kumarfbs@iitr.ernet.in; Sharma, Ashwani K., E-mail: kumarfbs@iitr.ernet.in [Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667 (India)

    2007-04-01

    A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. The crystals belong to the tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 75.8, c = 150.9 Å. The crystals contain two molecules in the asymmetric unit with a V{sub M} value of 2.5 Å{sup 3} Da{sup −1}. Diffraction was observed to 2.65 Å resolution and a complete data set was collected to 2.9 Å resolution.

  4. Trypsin inhibitory activity in blood and cervical mucus of sheep following chronic gamma irradiation

    International Nuclear Information System (INIS)

    Molnarova, M.; Arendarcik, J.; Pastorova, B.

    1982-01-01

    The effect was investigated of chronic gamma irradiation for a period of 7 days to a total dose of 6.7 Gy. A decrease was found in TIA (trypsin inhibition activity) of blood plasma to 73.6% of the value prior to irradiation. The low-molecular fraction of TIA increased to 194.6%. Later in the experiment the values decreased. The TIA dynamics of the cervical mucus had the reverse character. The values increased; at the 16th day after irradiation they increased up to 392% of the initial values. A comparison with previous experiments shows that acute local irradiation with an almost three-fold dose of the hypothalamo-pituitary area and of the ovaries does not induce significant changes in the blood plasma TIA. (M.D.)

  5. Monitoring of morphology and physical properties of cultured cells using a micro camera and a quartz crystal with transparent indium tin oxide electrodes after injections of glutaraldehyde and trypsin

    International Nuclear Information System (INIS)

    Kang, Hyen-Wook; Ida, Kazumi; Yamamoto, Yuji; Muramatsu, Hiroshi

    2008-01-01

    For investigating the effects of chemical stimulation to cultured cells, we have developed a quartz crystal sensor system with a micro charge-coupled device (CCD) camera that enables microphotograph imaging simultaneously with quartz crystal measurement. Human hepatoma cell line (HepG2) cells were cultured on the quartz crystal through a collagen film. The electrode of the quartz crystal was made of indium tin oxide (ITO) transparent electrodes that enable to obtain a transparent mode photograph. Glutaraldehyde and trypsin were injected to the chamber of the cells, respectively. The response of the quartz crystal was monitored and microphotographs were recorded, and the resonance frequency and resonance resistance were analyzed with an F-R diagram that plotted the resonance frequency and resonance resistance. In the case of the glutaraldehyde injection, the cells responded in two steps that included the fast response of the cross-linking reaction and the successive internal change in the cells. In the case of the trypsin injection, the responses included two processes. In the first step, cell adhesion factors were cleaved and the cell structure became round, and in the next step, the cells were deposited on the quartz crystal surface and the surface of the cells was directly in contact with the quartz crystal surface

  6. Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor.

    Science.gov (United States)

    Roelofs, Maarke J E; van den Berg, Martin; Bovee, Toine F H; Piersma, Aldert H; van Duursen, Majorie B M

    2015-03-02

    Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 μM, 60 μM, and 22 nM for GR, and 39 μM, 20 μM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular Δ4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Molecular mechanisms and in vitro antioxidant effects of Lactobacillus plantarum MA2.

    Science.gov (United States)

    Tang, Wei; Xing, Zhuqing; Li, Chao; Wang, Jinju; Wang, Yanping

    2017-04-15

    Lactobacillus plantarum MA2 was isolated from Chinese traditional Tibetan kefir grains. The antioxidant activities in vitro of this strain were evaluated extensively. The results showed that L. plantarum MA2 can tolerate hydrogen peroxide up to 2.0mM, and its fermentate (fermented supernatant, intact cell and cell-free extract) had strong reducing capacities, lipid peroxidation inhibition capacities, Fe 2+ -chelating abilities, as well as various free radical scavenging capacities. Additionally, both the fermented supernatant and cell homogenate exhibited glutathione peroxidase activity and superoxide dismutase activity. In order to investigate the antioxidant mechanism of L. plantarum MA2 at the molecular level, eight antioxidant-related genes were identified, and further analyzed. Three groups of genes cat, gshR and npx, were found up-regulated under H 2 O 2 challenge. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine beta-trypsin.

    Science.gov (United States)

    Jaśkiewicz, A; Lis, K; Rózycki, J; Kupryszewski, G; Rolka, K; Ragnarsson, U; Zbyryt, T; Wilusz, T

    1998-10-02

    Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

  9. Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis

    NARCIS (Netherlands)

    F.F.G. Rommerts (Focko); S.R. King (Steven); P.N. Span (Paul)

    2001-01-01

    textabstractIn virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period

  10. Increase in physical activities in kindergarten children with cerebral palsy by employing MaKey-MaKey-based task systems.

    Science.gov (United States)

    Lin, Chien-Yu; Chang, Yu-Ming

    2014-09-01

    In this study, we employed Flash- and Scratch-based multimedia by using a MaKey-MaKey-based task system to increase the motivation level of children with cerebral palsy to perform physical activities. MaKey MaKey is a circuit board that converts physical touch to a digital signal, which is interpreted by a computer as a keyboard message. In this study, we used conductive materials to control this interaction. This study followed single-case design using ABAB models in which A indicated the baseline and B indicated the intervention. The experiment period comprised 1 month and a half. The experimental results demonstrated that in the case of two kindergarten children with cerebral palsy, their scores were considerably increased during the intervention phrases. The developmental applications of the results are also discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis.

    NARCIS (Netherlands)

    Rommerts, F.F.; King, S.R.; Span, P.N.

    2001-01-01

    In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then

  12. Cellular cooperation in lymphocyte activation. III. B-cell helper effect in the enhancement of T-cell response.

    Science.gov (United States)

    Kasahara, T; Kin, K; Itoh, Y; Kawai, T; Kano, Y; Shioiri-Nakano, K

    1979-01-01

    T and B cells were purified from human tonsil and peripheral blood by the removal of phagocytic cells, followed by filtration through a nylon fiber column (NC) and E-rosette formation. Purified T and B cells contained less than 1% of other cell types. The responses of T cells to concanavalin A (Con A) and soluble protein A were greatly enhanced in the presence of autologous B cells. Participation of B cells in T-cell enhancement was confirmed by the following observations: (a) purified B copulation, which was separated further from adherent B cells, retained its enhancing activity. (b) Another adherent cell-free B-cell preparation, which was purified from the NC-passed fraction, and (c) no T lymphoid but some B lymphoid cell lines, elicited strong T-cell enhancement. It was also found that the enhancing capacity of B cells required no metabolic activity, but rather an intact cell form and direct cell-to-cell contact with responding cells. The stimulatory determinants on B cells were resistant to trypsin and neuraminidase treatment. In this paper a hypothesis will be presented that at least two signals are prerequisite for the effective activation of T cells.

  13. SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

    Science.gov (United States)

    Bak, Nicola; Rajagopal, Shalini; Stickings, Paul; Sesardic, Dorothea

    2017-07-20

    Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

  14. Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

    Science.gov (United States)

    França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

    2016-10-15

    A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.

    Science.gov (United States)

    Oparin, Peter B; Mineev, Konstantin S; Dunaevsky, Yakov E; Arseniev, Alexander S; Belozersky, Mikhail A; Grishin, Eugene V; Egorov, Tsezi A; Vassilevski, Alexander A

    2012-08-15

    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  16. Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

    Directory of Open Access Journals (Sweden)

    Alexandre Martins Costa Santos

    2008-08-01

    Full Text Available The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

  17. Crystallization, data collection and processing of the chymotrypsin–BTCI–trypsin ternary complex

    Energy Technology Data Exchange (ETDEWEB)

    Esteves, Gisele Ferreira; Teles, Rozeni Chagas Lima; Cavalcante, Nayara Silva; Neves, David; Ventura, Manuel Mateus [Laboratório de Biofísica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília-DF (Brazil); Barbosa, João Alexandre Ribeiro Gonçalves, E-mail: joao@lnls.br [Center for Structural Molecular Biology (CeBiME), Brazilian Synchrotron Light Laboratory (LNLS), CP 6192, 13083-970 Campinas-SP (Brazil); Freitas, Sonia Maria de, E-mail: joao@lnls.br [Laboratório de Biofísica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília-DF (Brazil)

    2007-12-01

    A ternary complex of the proteinase inhibitor (BTCI) with trypsin and chymotrypsin was crystallized and its crystal structure was solved by molecular replacement. A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin–BTCI–chymotrypsin ternary complex to 2.7 Å resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI–trypsin binary complex (PDB code) and chymotrypsin (PDB code) as search models.

  18. Crystallization, data collection and processing of the chymotrypsin–BTCI–trypsin ternary complex

    International Nuclear Information System (INIS)

    Esteves, Gisele Ferreira; Teles, Rozeni Chagas Lima; Cavalcante, Nayara Silva; Neves, David; Ventura, Manuel Mateus; Barbosa, João Alexandre Ribeiro Gonçalves; Freitas, Sonia Maria de

    2007-01-01

    A ternary complex of the proteinase inhibitor (BTCI) with trypsin and chymotrypsin was crystallized and its crystal structure was solved by molecular replacement. A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin–BTCI–chymotrypsin ternary complex to 2.7 Å resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI–trypsin binary complex (PDB code) and chymotrypsin (PDB code) as search models

  19. Carbohydrate as covalent crosslink in human inter-alpha-trypsin inhibitor

    DEFF Research Database (Denmark)

    Jessen, T E; Faarvang, K L; Ploug, M

    1988-01-01

    The primary structure of inter-alpha-trypsin inhibitor is partially elucidated, but controversy about the construction of the polypeptide backbone still exists. We present evidence suggesting that inter-alpha-trypsin inhibitor represents a novel plasma protein structure with two separate polypept...... polypeptide chains covalently crosslinked only by carbohydrate (chondroitin sulphate)....

  20. The HectoMAP Cluster Survey. I. redMaPPer Clusters

    Science.gov (United States)

    Sohn, Jubee; Geller, Margaret J.; Rines, Kenneth J.; Hwang, Ho Seong; Utsumi, Yousuke; Diaferio, Antonaldo

    2018-04-01

    We use the dense HectoMAP redshift survey to explore the properties of 104 redMaPPer cluster candidates. The redMaPPer systems in HectoMAP cover the full range of richness and redshift (0.08 systems included in the Subaru/Hyper Suprime-Cam public data release are bona fide clusters. The median number of spectroscopic members per cluster is ∼20. We include redshifts of 3547 member candidates listed in the redMaPPer catalog whether they are cluster members or not. We evaluate the redMaPPer membership probability spectroscopically. The purity (number of real systems) in redMaPPer exceeds 90% even at the lowest richness. Three massive galaxy clusters (M ∼ 2 × 1013 M ⊙) associated with X-ray emission in the HectoMAP region are not included in the public redMaPPer catalog with λ rich > 20, because they lie outside the cuts for this catalog.

  1. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

    Science.gov (United States)

    Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

    2014-10-01

    Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

  2. Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state

    Energy Technology Data Exchange (ETDEWEB)

    Blankenship, Elise; Vukoti, Krishna [Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Miyagi, Masaru, E-mail: mxm356@cwru.edu [Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Lodowski, David T., E-mail: mxm356@cwru.edu [Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States)

    2014-03-01

    This work reports the first sub-angstrom resolution structure of S. erythraeus trypsin. The detailed model of a prototypical serine protease at a catalytically relevant pH with an unoccupied active site is presented and is compared with other high-resolution serine protease structures. With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

  3. Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state

    International Nuclear Information System (INIS)

    Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T.

    2014-01-01

    This work reports the first sub-angstrom resolution structure of S. erythraeus trypsin. The detailed model of a prototypical serine protease at a catalytically relevant pH with an unoccupied active site is presented and is compared with other high-resolution serine protease structures. With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity

  4. Biological activity of Serratia marcescens cytotoxin

    Directory of Open Access Journals (Sweden)

    G.V. Carbonell

    2003-03-01

    Full Text Available Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 µg/ml of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.

  5. Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum).

    Science.gov (United States)

    Shamsi, Tooba Naz; Parveen, Romana; Sen, Priyankar; Fatima, Sadaf

    2017-05-28

    The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20 mM tris-CaCl 2 buffer (pH 8.2) with a flow rate of 0.5 mL min -1 . The molecular weight and purity of ∼23 kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697 U mg -1 , fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.

  6. Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crab Cancer pagurus

    International Nuclear Information System (INIS)

    Hehemann, Jan-Hendrik; Redecke, Lars; Perbandt, Markus; Saborowski, Reinhard; Betzel, Christian

    2007-01-01

    Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution. The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 Å resolution, respectively. The crystal of C.p.TryII belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 Å. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P2 1 3 with unit-cell parameters a = b = c = 215.4 Å, are assumed to contain 12 molecules per asymmetric unit

  7. The use of trypsin-like blood activity as a marker of pulmonary fibrosis severity.

    Directory of Open Access Journals (Sweden)

    V. V. Rodionova

    2018-05-01

    Full Text Available Purpose – to determine changes in the trypsin-like blood activity and its relationship with acute phase indices of inflammation in patients with pulmonary fibrosis (PF as a marker of course severity and prognosis of the disease. Materials and Methods: The study included 18 patients: 15 (83% women and 3 (17% men, mean age 53±2.5 years. The control group included 15 practically healthy persons. All the examined patients (n=18 were divided into two groups: with mild or moderately severe PF – 8 (44.4% patients (group I, severe PF – 10 (55.6% of patients (group II. Duration of the disease - from 1 month. up to 4 years. Patients underwent clinic-laboratory and anthropometric studies, a mMRC questionnaire was used, blood saturation was measured, lung radiography in 2 projections, echocardiography, and if necessary a high-resolution computer tomography etc were performed. Results and discussion: More than half of the patients were overweight (44,4% or had obesity of І-ІІІ st. (27.8%. The severity of dyspnea according to mMRC scale was 3.0 (3.0-4.0 points in patients of group II and 2.5 (2.0-3.0 points in patients of group I. There was a decrease in C-reactive protein (CRP in group I and a tendency to increase in patients of group II. When analyzing the indicator of trypsin-like blood activity (TLA, it was found that the median TLA at the beginning of the observation was twice as high as in healthy individuals, direct correlation was established between the level of TLA and the severity of the disease course. After treatment the level of TLA decreased in group I patients. In the severe course of PF, the average TLA level remained high. The progredient course of LF is characterized by severe clinical symptoms, a significant increase in TLA, CRP, reduced O2 saturation, a lack of response to treatment and an unfavorable prognosis. The TLA index can be used as a biochemical marker of the severity of PF along with the CRPindex, O2 saturation and

  8. Outer capsid proteins induce the formation of pores in epithelial cells

    International Nuclear Information System (INIS)

    Ruiz, M; Abad M; Michelangely, F; Charpilienne, A; Cohen, J

    1995-01-01

    Two mechanisms of entrance in cell of the rotavirus, during the infection, were proposed: a direct entrance through the plasmatic membrane or by means of endocytosis. In the two cases, a permeabilization mechanism of the membrane (cellular or of the endocytic vesicle, respectively) should occur. It has been shown that the rotavirus induces permeabilization of liposomes and of membrane vesicles. In this work, are studied the changes of intact cells permeability, measuring the entrance of e tide bromides. Viral particles of double capsid of the RF stump produce an increase of the cells membrane MA104 permeability, while the simple capsid ones don't induce effect. This phenomenon requires the particles trypsinization, and occurs in a means where the concentration of free Ca is lower to 1 micromolar. The temporary course of the fluorescence increase is sigmoid. The latency, the speed and the width depend on the relationship of virus / cell, and it can be observed up to 100% of permeabilization in relation to the effect of digitonin. The pores induced in the membrane by the rotavirus are irreversible. The permeabilizer effect of the rotavirus on the membrane was observed in other cellular lines as Hela and HT29, but not in the L929 ones. These results suggest that one or more proteins of the external capsid are responsible s of the effect. These could be involved in the penetration process of the virus towards the cytoplasm and could be one of the restrictive factor of the cell infection by means of the virus [es

  9. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    International Nuclear Information System (INIS)

    Bulleit, R.F.; Zimmerman, E.F.

    1984-01-01

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity

  10. Effect of trypsin inhibitor activity in soya bean on growth performance, protein digestibility and incidence of sub-clinical necrotic enteritis in broiler chicken flocks.

    Science.gov (United States)

    Palliyeguru, M W C D; Rose, S P; Mackenzie, A M

    2011-06-01

    1. The effect of three different levels of dietary trypsin inhibitor activity (achieved by varying the amount of non-toasted full fat soya bean in replacement for toasted full fat soya bean) on the incidence of spontaneously-occurring sub-clinical necrotic enteritis (NE) in broiler chickens was compared. A fourth dietary treatment compared the effect of a diet that used potato protein concentrate as the major protein source. The determined trypsin inhibitor activity increased with the increasing content of non-toasted soya bean: 1·90, 6·21, 8·46 and 3·72 mg/g for the three soya bean diets (0, 100 and 200 g of non-toasted soya bean/kg) and the potato protein diet respectively. 2. Although increasing amounts of the non-toasted full-fat soya bean increased the feed intakes of the birds, there was a marked reduction in protein digestibility, weight gain and feed conversion efficiency. 3. There was a linear increase in sub-clinical NE lesions in the duodenum, jejunum, mid small intestine and ileum with increasing non-toasted soya bean. Caecal Clostridium perfringens counts increased with the increasing dietary content of non-toasted soya bean. Serum α-toxin antibodies were higher in the birds fed the 200 g non-toasted soya bean/kg diet compared with the other diets. 4. The results demonstrated that variation in the amount of non-toasted dietary soya bean not only affects growth performance of broilers but also affects the incidence of sub-clinical necrotic enteritis in the flock. Ensuring the lowest possible trypsin-inhibitor activity in soya bean samples is a valuable tool to improve the health and welfare of birds and in reducing the financial losses from this disease.

  11. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    Science.gov (United States)

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

  12. Radiation hardening and irradiation testing of in-cell electronics for MA23/APM

    International Nuclear Information System (INIS)

    Friant, A.

    1988-09-01

    We relate briefly the radiation hardening method used to guarantee a gamma resistance of 10 Mrad for the whole electronic equipment associated with the slave arm of MA23 M servomanipulator which will be set up in cell 404 in Marcoule (APM). We describe the radiation testing of electronic devices and of the various subsystems designed by the D. LETI groups involved in the MA23/APM project

  13. Characterization of trypsin-derived peptides acrylamide-adducted hemoglobin

    International Nuclear Information System (INIS)

    Springer, D.L.; Goheen, S.C.; Edmonds, C.G.; McCulloch, M.; Sylvester, D.M.; Sander, C.; Bull, R.J.

    1991-01-01

    Even though there are a number of sources for human exposure to acrylamide, reliable biomarkers of exposure are not available. In an effort to develop such a biomarker, the authors are characterizing peptides derived from trypsin digests of acrylamide-adducted hemoglobin. For this, radiolabeled acrylamide was incubated with this, radiolabeled acrylamide was incubated with purified human hemoglobin (Ao) and decomposition products removed by dialysis. When the adducted hemoglobin was separated by reverse-phase HPLC, radioactivity eluted with the α and β subunits, suggesting covalent binding. Digestion of individual subunits with trypsin followed by reverse phase HPLC, indicated that most of the radioactivity associated with the α subunit co-eluted with a single peptide. Similar results were observed for the β subunit except that significant amounts of radioactivity eluted with the solvent front, suggesting that radioactivity was released by trypsin digestion. Currently, these preparation are under further characterization by electrospray ionization mass spectrometry. This approach will aid in the identification of the adducted will aid in the identification of the adducted peptide and subsequent preparation of an acrylamide-specific antibody

  14. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    Directory of Open Access Journals (Sweden)

    Roberto Franco Teixeira Corrêa

    Full Text Available Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ, resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7 when tested at 20 µg/mL.

  15. MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum.

    Science.gov (United States)

    Jin, Kai; Ming, Yue; Xia, Yu Xian

    2012-12-01

    Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.

  16. Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist

    Directory of Open Access Journals (Sweden)

    Wenbo Sai

    2017-03-01

    Full Text Available Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1, an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

  17. Enhancing MA transmutation by irradiation of (MA, Zr)Hx in FBR blanket region - 5383

    International Nuclear Information System (INIS)

    Konashi, K.; Ikeda, K.; Itoh, K.; Hirai, M.; Koyama, T.; Kurosaki, K.

    2015-01-01

    Minor actinide (MA) hydride is proposed as transmutation target in sodium-cooled mixed oxide fuelled fast reactor. Preliminarily calculations have been done to check the transmutation efficiency of MA hydride targets. Three different types of MA target, MA-Zr alloy, (MA, Zr)O 2 and (MA, Zr)H x , have been compared on MA transmutation rate. The targets are assumed to be loaded around an active core in a 280 MWe sodium-cooled reactor; 54 MA target assemblies are respectively arranged in a row in the radial blanket zone. They are supposed to be irradiated for one year and then be cooled for 60 days. The transmuted mass has been evaluated by three-dimensional diffusion calculation to be 25, 15, 61 kg/EFPY for the alloy, the oxide and the hydride respectively, where production of MA in the active core is taken into account. The transmutation mass by (MA, Zr)H x is much larger than those by the other types of targets, while the core characteristics remain sound by locating MA targets outside of the active core. On top of that, two kinds of (MA, Zr)O 2 targets which are combined with ZrH x (x=1.7) pins have been calculated. Major Research/Development items are selected to establish the MA hydride transmutation method by reviewing technologies applicable to the transmutation system. The practical use of the MA hydride transmutation method is not far ahead technically, since this method can be developed by the extension of existing technologies. (authors)

  18. Pediatric peripheral blood progenitor cell collection: haemonetics MCS 3P versus COBE Spectra versus Fresenius AS104.

    Science.gov (United States)

    Bambi, F; Faulkner, L B; Azzari, C; Gelli, A M; Tamburini, A; Tintori, V; Lippi, A A; Tucci, F; Bernini, G; Genovese, F

    1998-01-01

    An increasing number of apheresis machines are becoming available for peripheral blood progenitor cell (PBPC) collection in children. At the Children's Hospital of Florence (Italy), three apheresis machines were evaluated: MCS 3P (Haemonetics) (10 procedures in 4 patients, aged 10-12 years, weight 23.5-64 kg), Spectra, (COBE) (8 procedures in 3 patients, aged 4-17 years, weight 19-59 kg), and AS104 (Fresenius) (24 procedures in 9 patients, aged 2-16 years, weight 13.6-60 kg). For PBPC quantitative analysis, CD34 cytofluorimetry was employed. Relevant variables analyzed included efficiency of CD34+ cell extraction and enrichment, mononuclear cell purity and red cell contamination of the apheresis components, and platelet count decreases after leukapheresis. No significant differences in CD34+ cell-extraction abilities were found. However, the AS104 provided consistently purer leukapheresis components in terms of mononuclear cell and CD34+ cell enrichment (441 +/- 59%, vs. 240 +/- 35% and 290 +/- 42% for MCS 3P and Spectra, respectively). Postapheresis platelet counts dropped the least with the AS104. The smallest patient who underwent apheresis with MCS 3P (the only machine working on discontinuous flow and hence with greater volume shifts) weighed 23.5 kg and tolerated the procedure well, with no signs of hemodynamic instability. No significant complications were observed. All machines seem to have comparable PBPC extraction efficiency, but the AS104 seems to give the component with the greatest PBPC enrichment. This feature might be relevant for further ex vivo cell processing (CD34+ cell selection, expansion, and so on).

  19. Comparison of various molecular forms of bovine trypsin: Correlation of infrared spectra with X-ray crystal structures

    Energy Technology Data Exchange (ETDEWEB)

    Prestrelski, S.J. (Mount Sinai School of Medicine of the City Univ. of New York (USA)); Byler, D.M. (U.S. Department of Agriculture, Philadelphia, PA (USA)); Liebman, M.N. (AMOCO Technology Corporation, Naperville, IL (USA))

    1991-01-01

    Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm{sup {minus}1}) which arises from the backbone C{double bond}O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformation types and component bands in the amide I region. In this paper the authors report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain ({beta}-trypsin) and once-cleaved, double-chain ({alpha}-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and {beta}-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. The spectroscopic results from these proteins in D{sub 2}O imply that certain loop structures may absorb in the region of 1655 cm{sup {minus}1}. They estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.

  20. The Drosophila KIF1A homolog unc-104 is important for site-specific active zone maturation

    Directory of Open Access Journals (Sweden)

    Yao V. Zhang

    2016-09-01

    Full Text Available Abstract Mutations in the kinesin-3 family member KIF1A have been associated with hereditary spastic paraplegia, hereditary sensory and autonomic neuropathy type 2 and intellectual disability. Both autosomal recessive and autosomal dominant forms of inheritance have been reported. Loss of KIF1A or its homolog unc-104 causes early postnatal or embryonic lethality in mice and Drosophila, respectively. In this study we use a previously described hypomorphic allele of unc-104, unc-104bris, to investigate the impact of partial loss-of-function of kinesin-3 function on active zone formation at the Drosophila neuromuscular junction. unc-104bris mutants exhibit synaptic defects where a subset of synapses at the neuromuscular junction lack the key active zone organizer protein Bruchpilot. Modulating synaptic Bruchpilot levels by ectopic overexpression or RNAi-mediated knockdown suggests that the loss of active zone components such as Ca2+ channel and Liprin-α from these synapses is caused by impaired kinesin-3 transport rather than due to the absence of Bruchpilot at these synapses. In addition to defects in active zone maturation, unc-104bris mutants display impaired transport of dense core vesicles and synaptic vesicle associated proteins, among which Rab3 has been shown to regulate the distribution of Bruchpilot to active zones. Overexpression of Rab3 partially ameliorates synaptic phenotypes of unc-104bris neuromuscular junction, suggesting that lack of presynaptic Rab3 may contribute to defects in synapse maturation.

  1. Antibodies from malaria-exposed pregnant women recognize trypsin resistant epitopes on the surface of Plasmodium falciparum-infected erythrocytes selected for adhesion to chondroitin sulphate A

    DEFF Research Database (Denmark)

    Sharling, Lisa; Enevold, Anders; Sowa, Kordai M P

    2004-01-01

    of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved.......-specific antibodies induced as a result of pregnancy associated malaria (PAM). METHODS: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P....... falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved...

  2. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  3. Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus).

    Science.gov (United States)

    Liu, Zi-Qiang; Tuo, Feng-Yan; Song, Liang; Liu, Yu-Xin; Dong, Xiu-Ping; Li, Dong-Mei; Zhou, Da-Yong; Shahidi, Fereidoon

    2018-08-01

    Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Trypsin diminishes the rat potency of polio serotype 3.

    Science.gov (United States)

    ten Have, R; Westdijk, J; Levels, L M A R; Koedam, P; de Haan, A; Hamzink, M R J; Metz, B; Kersten, G F A

    2015-11-01

    This study addresses observations made in view of testing in practice the guideline in the European Pharmacopoeia (EP) on omitting the rat potency test for release of polio containing vaccines. In general, use of the guideline is valid and the D-antigen ELISA can indeed be used as an in vitro alternative for the in vivo test. However, the set-up of the ELISA is crucial and should include detection of antigenic site 1 in polio serotype 3 as destruction of that site by trypsin results in a reduced rat potency. Antigenic site 1 in polio serotype 2 may also be modified by trypsin, but the cleavage of viral protein 1 (VP1) did not affect the rat potency. Therefore, any antigenic site, except site 1, can be used for detection of polio serotype 2. It is advised to include testing of the effect of trypsin treatment in the EP-guideline. This allows polio vaccine manufacturers to check whether their in-house ELISA needs improvement. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  5. 50 CFR 224.104 - Special requirements for fishing activities to protect endangered sea turtles.

    Science.gov (United States)

    2010-10-01

    ... activities to protect endangered sea turtles. 224.104 Section 224.104 Wildlife and Fisheries NATIONAL MARINE... endangered sea turtles. (a) Shrimp fishermen in the southeastern United States and the Gulf of Mexico who comply with rules for threatened sea turtles specified in § 223.206 of this chapter will not be subject...

  6. Structure basis 1/2SLPI and porcine pancreas trypsin interaction

    Energy Technology Data Exchange (ETDEWEB)

    Fukushima, Kei; Kamimura, Takashi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Institute for Bio-Medical Research, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

    2013-11-01

    1/2SLPI is a C-terminal domain of SLPI (secretory leukocyte protease inhibitor) which inhibits various serine proteases broadly. The present study is the first X-ray structural report on how 1/2SLPI with P1 Leu strongly inhibits trypsin and how it can inhibit multiple serine proteases. SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58–Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2′ to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

  7. Regulation of the fate of dental-derived mesenchymal stem cells using engineered alginate-GelMA hydrogels.

    Science.gov (United States)

    Ansari, Sahar; Sarrion, Patricia; Hasani-Sadrabadi, Mohammad Mahdi; Aghaloo, Tara; Wu, Benjamin M; Moshaverinia, Alireza

    2017-11-01

    Mesenchymal stem cells (MSCs) derived from dental and orofacial tissues provide an alternative therapeutic option for craniofacial bone tissue regeneration. However, there is still a need to improve stem cell delivery vehicles to regulate the fate of the encapsulated MSCs for high quality tissue regeneration. Matrix elasticity plays a vital role in MSC fate determination. Here, we have prepared various hydrogel formulations based on alginate and gelatin methacryloyl (GelMA) and have encapsulated gingival mesenchymal stem cells (GMSCs) and human bone marrow MSCs (hBMMSCs) within these fabricated hydrogels. We demonstrate that addition of the GelMA to alginate hydrogel reduces the elasticity of the hydrogel mixture. While presence of GelMA in an alginate-based scaffold significantly increased the viability of encapsulated MSCs, increasing the concentration of GelMA downregulated the osteogenic differentiation of encapsulated MSCs in vitro due to decrease in the stiffness of the hydrogel matrix. The osteogenic suppression was rescued by addition of a potent osteogenic growth factor such as rh-BMP-2. In contrast, MSCs encapsulated in alginate hydrogel without GelMA were successfully osteo-differentiated without the aid of additional growth factors, as confirmed by expression of osteogenic markers (Runx2 and OCN), as well as positive staining using Xylenol orange. Interestingly, after two weeks of osteo-differentiation, hBMMSCs and GMSCs encapsulated in alginate/GelMA hydrogels still expressed CD146, an MSC surface marker, while MSCs encapsulated in alginate hydrogel failed to express any positive staining. Altogether, our findings suggest that it is possible to control the fate of encapsulated MSCs within hydrogels by tuning the mechanical properties of the matrix. We also reconfirmed the important role of the presence of inductive signals in guiding MSC differentiation. These findings may enable the design of new multifunctional scaffolds for spatial and temporal

  8. Preparation and characterization of magnetic levan particles as matrix for trypsin immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Maciel, J.C. [Programa de Pos-Graduacao em Ciencias Biologicas, Universidade Federal de Pernambuco, Cidade Universitaria, 50670-901 Recife, PE (Brazil); Andrad, P.L. [Programa de Pos-Graduacao em Ciencia de Materiais, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Neri, D.F.M., E-mail: davidfmneri@yahoo.com.br [Universidade Federal do Vale do Sao Francisco, 56304-205 Petrolina, PE (Brazil); Carvalho, L.B. [Departamento de Bioquimica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Cardoso, C.A. [Departamento de Fisica, Universidade Federal de Sao Carlos, 13565-905 Sao Carlos, PE (Brazil); Calazans, G.M.T. [Departamento de Antibioticos, Universidade Federal de Pernambuco, Cidade Universitaria, 50670-901 Recife, PE (Brazil); Albino Aguiar, J. [Departamento de Fisica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil); Silva, M.P.C. [Departamento de Bioquimica, Universidade Federal de Pernambuco, Cidade Universitaria, 50679-901 Recife, PE (Brazil)

    2012-04-15

    Magnetic levan was synthesized by co-precipitating D-fructofuranosyl homopolysaccharide with a solution containing Fe{sup 2+} and Fe{sup 3+} in alkaline conditions at 100 Degree-Sign C. The magnetic levan particles were characterized by scanning electron microscopy (SEM), magnetization measurements, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Afterwards, magnetic levan particles were functionalized by NaIO{sub 4} oxidation and used as matrices for trypsin covalent immobilization. Magnetite and magnetic levan particles were both heterogeneous in shape and levan-magnetite presented bigger sizes compared to magnetite according to SEM images. Magnetic levan particles exhibited a magnetization 10 times lower as compared to magnetite ones, probably, due to the coating layer. XRD diffractogram showed that magnetite is the dominant phase in the magnetic levan. Infrared spectroscopy showed characteristics absorption bands of levan and magnetite (O-H, C-O-C and Fe-O bonds). The immobilized trypsin derivative was reused 10 times and lost 16% of its initial specific activity only. Therefore, these magnetic levan particles can be proposed as an alternative matrices for enzyme immobilization. - Highlights: Black-Right-Pointing-Pointer The magnetic levan particles presented larger size variation than magnetite particles due to the changes produced by coating. Black-Right-Pointing-Pointer The utilization of magnetic levan particles showed to be efficacious for immobilization of enzymes as trypsin. Black-Right-Pointing-Pointer Magnetic particles can be planned as other matrix for immobilization of biomolecule in various division processes in biotechnology.

  9. High feline trypsin-like immunoreactivity in a cat with pancreatitis and hepatic lipidosis.

    Science.gov (United States)

    Bruner, J M; Steiner, J M; Williams, D A; Van Alstine, W G; Blevins, W

    1997-06-15

    A 1.5-year-old domestic shorthair cat was examined because of vomiting and icterus. Clinicopathologic abnormalities included high alanine transaminase, alkaline phosphatase, and gamma-glutamyltransferase activities and high total bilirubin concentration. During abdominal ultrasonography, the left limb and body of the pancreas appeared hypoechoic, and a small quantity of peritoneal effusion was seen. The liver was diffusely hyperechoic, with echogenicity similar to that of the spleen, indicating hepatic lipidosis. Feline trypsin-like immunoreactivity was high, suggesting that the cat also had pancreatitis. The cat was treated with crystalloid fluids and was fed a protein-restricted diet via a percutaneous endoscopically placed gastrostomy tube. The cat's condition continued to deteriorate despite medical treatment, and it was euthanatized. Necropsy confirmed the clinical suspicion of acute pancreatitis and hepatic lipidosis. This case suggests that measurement of trypsin-like immunoreactivity may be useful in cats suspected of having pancreatitis.

  10. 34 CFR 104.1 - Purpose.

    Science.gov (United States)

    2010-07-01

    ... NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 104.1 Purpose. The purpose of this part is to effectuate section 504 of the Rehabilitation Act... 34 Education 1 2010-07-01 2010-07-01 false Purpose. 104.1 Section 104.1 Education Regulations of...

  11. Karakterisasi stem cell pulpa gigi sulung dengan modifikasi enzim tripsin (The characterization of stem cells from human exfoliated deciduous teeth using trypsin enzym

    Directory of Open Access Journals (Sweden)

    Tri Wijayanti Puspitasari

    2014-06-01

    Full Text Available Background: Now a days, treatment in dentistry, using tissue regeneration that based on the stem cells from human exfoliated deciduous teeth (SHED, grows rapidly. For several reason, the isolated and cultured SHED is difficult to be applied in Indonesia, therefore the modification is needed. This difficulties were caused by the pulp anatomy, the heterogeneous populations in the pulp chamber and the limitations of tools and materials at the laboratory. Purpose: This research was aimed to examine that the modifications of isolation and culture technique of SHEDs for characterization by using the marker of CD105. Methods: The research was experimental laboratory with the cross sectional design. The samples were the human exfoliated deciduous teeth from the children patients of Pediatric Dentistry Department of Universitas Airlangga Dental Hospital which matched the criteria. Dental pulps were isolated and cultured by using the modifications of Trypsin enzymes. Results: The healthy SHEDs could be produced from the modifications of isolation and culture and positively shown the expression of marker CD105 which were indicated by the fluorencent microscope. Conclusion: SHED which isolated and cultured by using the modified techniques, positively characterized by using marker CD105.Latar Belakang: Pengobatan kedokteran gigi berkembang dengan pesat terutama di bidang regenerasi jaringan berbasis Stem Cells from Human Exfoliated Deciduous Teeth (SHED. Di Indonesia, isolasi dan kultur SHED sulit sehingga perlu dilakukan modifikasi. Kendala ini muncul karena jaringan pulpa yang kecil, heterogen dan keterbatasan alat dan bahan di laboratorium. Tujuan: Penelitian ini bertujuan untuk meneliti modifikasi pada cara isolasi dan kultur SHED untuk karakterisasi menggunakan maker CD105. Metode: Jenis penelitian ini adalah eksperimental laboratoris dengan rancangan cross sectional. Sampel penelitian adalah gigi sulung dari pasien anak di Klinik Kedokteran Gigi Anak

  12. Physical-chemical characterization and stability study of alpha-trypsin at ph 3.0 by differential scanning calorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Santos, A.M.C.; Santana, M.A.; Gomide, F.T.F.; Oliveira, J.S.; Vilas Boas, F.A.S.; Santoro, M.M.; Teixera, K.N. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas (ICB). Dept. de Bioquimica e Imunologia; Miranda, A.A.C.; Biondi, I. [Universidade Estadual de Feira de Santana (UEFS), BA (Brazil). Dept. de Ciencias Biologicas; Vasconcelos, A.B.; Bemquerer, M.P. [EMBRAPA Recursos Geneticos e Biotecnologia, Brasilia, DF (Brazil). Parque Estacao Biologica (PqEB)

    2008-07-01

    Full text: {alpha}-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel {beta}-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of {alpha}-trypsin in the acid pH range was performed and physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The {alpha}-trypsin has a shelf-life (t{sub 95%}) of about ten months at pH 3.0 and 4 deg C and its hydrolysis into the {psi}-trypsin isoform is negligible during six months as monitored by mass spectrometry (Micromass Q-ToF). The observed {delta}H{sub cal}/{delta}H{sub vH} ratio is close to unity for {alpha}-trypsin denaturation, which suggests the occurrence of a two-state transition, devoid of molten-globule intermediates. At pH 3.0, {alpha}-trypsin unfolded with T{sub m} 325.9 K and {delta}H= 99.10 kcal mol{sup -1}, and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96 {+-} 0.18 kcal mol{sup -1} K{sup -1}. The stability of {alpha}-trypsin calculated at 298 K and at pH 3.0 was {delta}G{sub U} = 6.10 kcal mol{sup -1}. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters for unfolding of {beta}-trypsin do not change substantially after its conversion to {alpha}-trypsin.

  13. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  14. Correlating The Star Formation Histories Of MaNGA Galaxies With Their Past AGN Activity

    Science.gov (United States)

    Gonzalez Ortiz, Andrea

    2017-01-01

    We investigate active galactic nuclei (AGN) as a primary mechanism affecting star formation in MaNGA galaxies. Using the Pipe3D code, we modeled the stellar population from MaNGA spectra and derived the star formation histories of 53 AGN host galaxies. We seek to compare the star formation histories of the host galaxies of AGN with the ages of their radio lobes to better understand the role of AGN feedback in the star formation histories of MaNGA galaxies. MaNGA (Mapping Nearby Galaxies at APO) is one of the three core programs in the fourth generation Sloan Digital Sky Survey(SDSS). MaNGA will investigate the internal kinematics of nearly 10,000 local galaxies through dithered observations using fiber integral field units (IFUs) that vary in diameter from 12" (19 fibers) to 32" (127 fibers). In this poster, we present initial results on the star formation histories of MaNGA AGN host galaxies. This work was supported by the SDSS Research Experience for Undergraduates program, which is funded by a grant from Sloan Foundation to the Astrophysical Research Consortium.

  15. Serum trypsin inhibitory capacity in hemodialysis patients

    International Nuclear Information System (INIS)

    Hashemi, Mohammad; Mehrabifar, Hamid; Homayooni, Fatemeh; Naderi, Mohammad; Montazerifar, Farzaneh; Ghavami, Saeid

    2009-01-01

    It has been established that overproduction of reactive oxygen species (ROS) occurs during hemodialysis causing oxidation of proteins. Alpha-1-antitrypsin is the major circulating anti-protease which contains methionine in the active site. The aim of the present study was to measure the level of serum trypsin inhibitory capacity (sTIC) in hemodialysis patients. This case-control study was performed in 52 hemodialysis patients and 49 healthy controls. sTIC was measured by enzymatic assay. The sTIC was significantly (P< 0.001) lower in hemodialysis patients (1.87 + - 0.67 micron mol/min/mL) than healthy controls (2.83 + - 0.44 micron mol/min/L). Reduction of sTIC may be due to the oxidation of methionine residue in the reactive site of alpha-1 antitrypsin. (author)

  16. Structural and Chemical Characterization of Silica Spheres before and after Modification by Silanization for Trypsin Immobilization

    Directory of Open Access Journals (Sweden)

    Eduardo F. Barbosa

    2017-01-01

    Full Text Available In the last decades, silica particles of a variety of sizes and shapes have been characterized and chemically modified for several applications, from chromatographic separation to dental supplies. The present study proposes the use of aminopropyl triethoxysilane (APTS silanized silica particles to immobilize the proteolytic enzyme trypsin for the development of a bioreactor. The major advantage of the process is that it enables the polypeptides hydrolysis interruption simply by removing the silica particles from the reaction bottle. Silanized silica surfaces showed significant morphological changes at micro- and nanoscale level. Chemical characterization showed changes in elemental composition, chemical environment, and thermal degradation. Their application as supports for trypsin immobilization showed high immobilization efficiency at reduced immobilization times, combined with more acidic conditions. Indirect immobilization quantification by reversed-phase ultrafast high performance liquid chromatography proved to be a suitable approach due to its high linearity and sensitivity. Immobilized trypsin activities on nonmodified and silanized silica showed promising features (e.g., selective hydrolysis for applications in proteins/peptides primary structure elucidation for proteomics. Silanized silica system produced some preferential targeting peptides, probably due to the hydrophobicity of the nanoenvironment conditioned by silanization.

  17. Inhibitory effects of methamphetamine on mast cell activation and cytokine/chemokine production stimulated by lipopolysaccharide in C57BL/6J mice.

    Science.gov (United States)

    Xue, Li; Geng, Yan; Li, Ming; Jin, Yao-Feng; Ren, Hui-Xun; Li, Xia; Wu, Feng; Wang, Biao; Cheng, Wei-Ying; Chen, Teng; Chen, Yan-Jiong

    2018-04-01

    Previous studies have demonstrated that methamphetamine (MA) influences host immunity; however, the effect of MA on lipopolysaccharide (LPS)-induced immune responses remains unknown. Mast cells (MCs) are considered to serve an important role in the innate and acquired immune response, but it remains unknown whether MA modulates MC activation and LPS-stimulated cytokine production. The present study aimed to investigate the effect of MA on LPS-induced MC activation and the production of MC-derived cytokines in mice. Markers for MC activation, including cluster of differentiation 117 and the type I high affinity immunoglobulin E receptor, were assessed in mouse intestines. Levels of MC-derived cytokines in the lungs and thymus were also examined. The results demonstrated that cytokines were produced in the bone marrow-derived mast cells (BMMCs) of mice. The present study demonstrated that MA suppressed the LPS-mediated MC activation in mouse intestines. MA also altered the release of MC cytokines in the lung and thymus following LPS stimulation. In addition, LPS-stimulated cytokines were decreased in the BMMCs of mice following treatment with MA. The present study demonstrated that MA may regulate LPS-stimulated MC activation and cytokine production.

  18. Radio-immuno-assay for trypsin in newborn-screening for cystic fibrosis

    International Nuclear Information System (INIS)

    Sander, J.; Niehaus, C.

    1982-01-01

    In 4,956 infants the concentration of immunoreactive trypsin was measured in dried blood on filter paper using a double antibody radioimmuno assay. About 90% of all results were below 40 ng/ml. In 13 infants the concentration of immunoreactive trypsin exceeded 80 ng/ml. These infants were examined clinically, including sweationtophoresis. We found three children suffering from cystic fibrosis. One further child showing an elevated concentration of chloride in the sweat (60 mval/ml) could not be reexamined. The concentration of immunoreactive trypsin in the cystic fibrosis children was 230, 297, and in one case 108 ng/ml at the 76th day of life, whereas the values for the 9 other children were between 80 and 154 ng/ml. We believe these results justify to use this test for a much higher number of infants, especially because it is inexpensive and can easily be added to existing newborn screening programs for inborn errors of metabolism. (orig.) [de

  19. MOFzyme: Intrinsic protease-like activity of Cu-MOF

    Science.gov (United States)

    Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

    2014-10-01

    The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

  20. 34 CFR 104.21 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Discrimination prohibited. 104.21 Section 104.21... ASSISTANCE Accessibility § 104.21 Discrimination prohibited. No qualified handicapped person shall, because a... excluded from participation in, or otherwise be subjected to discrimination under any program or activity...

  1. Mechanisms involved in the chemical inhibition of the Eosin-sensitized photooxidation of trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Rizzuto, F.; Spikes, J.D.

    1975-01-01

    A large series of compounds was screened for ability to protect trypsin from eosin-sensitized photodynamic inactivation. Eosin-sensitized photooxidation reactions of this type typically proceed via the triplet state of the dye and often involve singlet state oxygen as the oxidizing entity. In order to determine the mechanisms by which trypsin is protected from photoinactivation, a number of good protective agents (inhibitors) and some non-protective agents were selected for more detailed flash photolysis studies. Good inhibitors such as p-phenylenediamine, n-propyl gallate, serotonin creatinine sulfate and p-toluenediamine competed efficiently with oxygen and with trypsin for reaction with the triplet state of eosin. The inhibitors were shown to quench triplet eosin to the ground state and/or reduce triplet eosin to form the semireduced eosin radical and an oxidized form of the inhibitor. In the latter case, oxidized inhibitor could react by a reverse electron transfer reaction with the semireduced eosin radical to regenerate ground state eosin and the inhibitor. The good inhibitors also competed effectively with trypsin for oxidation by semioxidized eosin, thus giving another possible protective mechanism. Non-inhibitors such as halogen ions and the paramagnetic ions Co/sup + +/, Cu/sup + +/ and Mn/sup + +/ reacted only slowly with triplet and with semioxidized eosin. The primary pathway for the eosin-sensitized photooxidation of trypsin at pH 8.0 involved singlet oxygen, although semioxidized eosin may also participate.

  2. Preliminary X-Ray Crystallographic Studies of the N-Terminal Domains of Hsp104 from Yeast Candida albicans and Saccharomyces cerevisiae

    Science.gov (United States)

    Wang, P.; Li, J.; Sha, B.

    2017-12-01

    Yeast Hsp104 is an ATP-dependent molecular chaperone, which can solublize and rescue denatured proteins from aggregates into active form by cooperating with Hsp70 and Hsp40 chaperones. Moreover, overexpression of Hsp104 of Saccharomyces cerevisiae (ScHsp104) cures the yeast [ PSI +] prion due to the completely dissolution of the prion seeds, demonstrating ScHsp104's potential to clear amyloid-like protein aggregates, thus making ScHsp104 a promising medication approach for human amyloidogenic neurodegenerative diseases. Because the working mechanisms for ScHsp104's activities have not been clearly elucidated yet, crystallographic determination of ScHsp104 stands for great significance. Here, the expression, purification and crystallization of the N-terminal domains of Hsp104 from yeast Candida albicans (CaHsp104N) and S. cerevisiae (ScHsp104N) are described. The CaHsp104N crystals diffracted to 1.54 Å and belonged to the sp. gr. P3221 or P3121, with unit cell parameters of a = 55.213 Å, c = 109.451 Å. The data of the ScHsp104N crystals were collected to the resolution of 2.53 Å in the sp. gr. C2, with unit cell parameters a = 148.587 Å, b = 66.255 Å, c = 74.577 Å, β = 107.369°. The phase of ScHsp104N is determined by the molecular replacement method using CaHsp104N as the search model.

  3. Production of high concentration of L-lactic acid from cellobiose by thermophilic Bacillus coagulans WCP10-4.

    Science.gov (United States)

    Ong, Shufen Angeline; Ng, Zhi Jian; Wu, Jin Chuan

    2016-07-01

    Thermophilic Bacillus coagulans WCP10-4 is found to be able to convert cellobiose to optically pure L-lactic acid. Its β-glucosidase activity is detected in whole cells (7.3 U/g dry cells) but not in culture medium, indicating the intracellular location of the enzyme. Its β-glucosidase activity is observed only when cultured using cellobiose as the sole carbon source, indicating that the expression of this enzyme is tightly regulated in cells. The enzyme is most active at 50 °C and pH 7.0. The supplement of external β-glucosidase during fermentation of cellobiose (106 g/l) by B. coagulans WCP10-4 increased the fermentation time from 21 to 23 h and decreased the lactic acid yield from 96.1 to 92.9 % compared to the control without β-glucosidase supplementation. B. coagulans WCP10-4 converted 200 g/l of cellobiose to 196.3 g/l of L-lactic acid at a yield of 97.8 % and a productivity of 7.01 g/l/h. This result shows that B. coagulans WCP10-4 is a highly efficient strain for converting cellobiose to L-lactic acid without the need of supplementing external β-glucosidases.

  4. Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

    Science.gov (United States)

    Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

    2015-01-01

    Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

  5. Rotavirus-Like Particles: A Novel Nanocarrier for the Gut

    Directory of Open Access Journals (Sweden)

    Naima G. Cortes-Perez

    2010-01-01

    Full Text Available The delivery of bioactive molecules directly to damaged tissues represents a technological challenge. We propose here a new system based on virus-like particles (VLP from rotavirus, with a marked tropism for the gut to deliver bio-active molecules to intestinal cells. For this, nonreplicative VLP nanoparticles were constructed using a baculovirus expression system and used to deliver an exogenous biomolecule, the green fluorescent protein (GFP, into either MA104 cells or intestinal cells from healthy and 2,4,6-trinitrobenzene sulfonic acid (TNBS-treated mice. Our results show that expression of rotavirus capsid proteins in baculovirus led to the auto assembly of VLP that display similar properties to rotavirus. In vitro experiments showed that VLP were able to enter into MA104 cells and deliver the reporter protein. Intragastric administration of fluorescent VLP in healthy and TNBS-treated mice resulted in the detection of GFP and viral proteins in intestinal samples. Our results demonstrate an efficient entry of non-replicative rotavirus VLP into the epithelial cell line MA104 and provide the first in vivo evidence of the potential of these nanoparticles as a promising safe candidate for drug delivery to intestinal cells.

  6. 34 CFR 104.22 - Existing facilities.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Existing facilities. 104.22 Section 104.22 Education... Accessibility § 104.22 Existing facilities. (a) Accessibility. A recipient shall operate its program or activity.... This paragraph does not require a recipient to make each of its existing facilities or every part of a...

  7. Elastase production by B16-F10 melanoma cells

    International Nuclear Information System (INIS)

    Shrager, J.B.; Yusa, T.; Netland, P.A.; Zetter, B.R.

    1986-01-01

    Elastolytic activity was found in sonicates of mouse B16-F10 melanoma cells and in medium conditioned by these cells. Degradation of elastin was determined by measuring the release of soluble 3 H-peptides from labelled insoluble elastin. The activity secreted from B16-F10 cells was soluble and was not associated with membrane vesicles. The secreted activity was partially inhibited by incubation with phenymethylsulfonylfluoride (PMSF) and was abolished by incubation with the alpha-1-protease inhibitor, with pepstatin A or with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). In contrast, the activity was unaffected by incubation with elastatinal, with the plasmin inhibitor Σ-aminocaproic acid (EACA), the metalloproteinase inhibitor ethylenediamine-tetra-acetic acid (EDTA), the soybean trypsin inhibitor or the trypsin inhibitor N proportional to-p-tosyl-L-lysine chloromethyl ketone (TLCK). These results suggest that the majority of the tumor cell-derived elastolytic activity is attributable to a serine protease that differs in specificity from the well characterized elastases previously isolated from neutrophils, macrophages or from mammalian pancreas. The authors postulate that the release of elastase from lung-colonizing B16-F10 cells may facilitate their invasion of elastin-rich lung tissue

  8. Laue diffraction as a tool in dynamic studies: Hydrolysis of a transiently stable intermediate in catalysis by trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Singer, P.T.; Berman, L.E.; Cai, Z.; Mangel, W.F.; Jones, K.W.; Sweet, R.M. (Brookhaven National Lab., Upton, NY (United States)); Carty, R.P. (State Univ. of New York, Brooklyn, NY (United States). Dept. of Biochemistry); Schlichting, I. (Brandeis Univ., Waltham, MA (United States). Rosenstiel Basic Medical Science Center); Stock, A. (Center for Advanced Biotechnology and Medicine, Piscataway, NJ (Un

    1992-01-01

    A transiently stable intermediate in trypsin catalysis, guanidinobenzyol-Ser-195 trypsin, can be trapped and then released by control of the pH in crystals of the enzyme. This effect has been investigated by static and dynamic white-beam Laue crystallography. Comparison of structures determined before and immediately after a pH jump reveals the nature of concerted changes that accompany activation of the enzyme. Careful analysis of the results of several structure determinations gives information about the reliability of Laue results in general. A study of multiple exposures taken under differing conditions of beam intensity, crystal quality, and temperature revealed information about ways to control damage of specimens by the x-ray beam.

  9. Laue diffraction as a tool in dynamic studies: Hydrolysis of a transiently stable intermediate in catalysis by trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Singer, P.T.; Berman, L.E.; Cai, Z.; Mangel, W.F.; Jones, K.W.; Sweet, R.M. [Brookhaven National Lab., Upton, NY (United States); Carty, R.P. [State Univ. of New York, Brooklyn, NY (United States). Dept. of Biochemistry; Schlichting, I. [Brandeis Univ., Waltham, MA (United States). Rosenstiel Basic Medical Science Center; Stock, A. [Center for Advanced Biotechnology and Medicine, Piscataway, NJ (United States); Smalas, A. [Univ. of Tromso (Norway). Inst. of Mathematics and Physical Science

    1992-11-01

    A transiently stable intermediate in trypsin catalysis, guanidinobenzyol-Ser-195 trypsin, can be trapped and then released by control of the pH in crystals of the enzyme. This effect has been investigated by static and dynamic white-beam Laue crystallography. Comparison of structures determined before and immediately after a pH jump reveals the nature of concerted changes that accompany activation of the enzyme. Careful analysis of the results of several structure determinations gives information about the reliability of Laue results in general. A study of multiple exposures taken under differing conditions of beam intensity, crystal quality, and temperature revealed information about ways to control damage of specimens by the x-ray beam.

  10. Laue diffraction as a tool in dynamic studies: Hydrolysis of a transiently stable intermediate in catalysis by trypsin

    International Nuclear Information System (INIS)

    Singer, P.T.; Berman, L.E.; Cai, Z.; Mangel, W.F.; Jones, K.W.; Sweet, R.M.; Carty, R.P.; Smalas, A.

    1992-01-01

    A transiently stable intermediate in trypsin catalysis, guanidinobenzyol-Ser-195 trypsin, can be trapped and then released by control of the pH in crystals of the enzyme. This effect has been investigated by static and dynamic white-beam Laue crystallography. Comparison of structures determined before and immediately after a pH jump reveals the nature of concerted changes that accompany activation of the enzyme. Careful analysis of the results of several structure determinations gives information about the reliability of Laue results in general. A study of multiple exposures taken under differing conditions of beam intensity, crystal quality, and temperature revealed information about ways to control damage of specimens by the x-ray beam

  11. Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples

    Science.gov (United States)

    Abad, F. Xavier; Pintó, Rosa M.; Bosch, Albert

    1998-01-01

    A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Itor P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 106 and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection. PMID:9647805

  12. JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment.

    Science.gov (United States)

    Costa, Helen P S; Oliveira, Jose T A; Sousa, Daniele O B; Morais, Janne K S; Moreno, Frederico B; Monteiro-Moreira, Ana Cristina O; Viegas, Ricardo A; Vasconcelos, Ilka M

    2014-01-01

    Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0~kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252~kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.

  13. JcTI-I, a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment

    Directory of Open Access Journals (Sweden)

    Helen Paula S Costa

    2014-01-01

    Full Text Available Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v followed by affinity chromatography (Trypsin-Sepharose 4B and molecular exclusion (Sephacryl S-200. Non-reducing SDS-PAGE and gel filtration showed that JcTI-I has approximately 20.0 kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252 kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration (MIC less than 5 µg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.

  14. Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun; Chang, Mun Seock; Lopez-Ferrer, Daniel; Smith, Richard D.; Gu, Man Bock; Lee, Sang-Won; Kim, Beom S.; Kim, Jungbae

    2010-12-15

    An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

  15. Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt.

    Science.gov (United States)

    Wei, Yunxie; Liu, Wen; Hu, Wei; Liu, Guoyin; Wu, Chunjie; Liu, Wei; Zeng, Hongqiu; He, Chaozu; Shi, Haitao

    2017-08-01

    MaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance against Foc in banana. Autophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

  16. Cytogenetic study of Ascaris trypsin inhibitor in cultured human ...

    Indian Academy of Sciences (India)

    2009-04-01

    Apr 1, 2009 ... Although the physical and chemical properties of Ascaris trypsin inhibitors ... male of Ascaris suum according to the method of Pudles and. Rola (1967). ..... inhibitor isolated from Ascaris resulted in the appearance of dominant ...

  17. A novel dianionic amino acid ionic liquid-coated PEG 4000 modified Fe3O4 nanocomposite for the magnetic solid-phase extraction of trypsin.

    Science.gov (United States)

    Yang, Qin; Wang, Yuzhi; Zhang, Hongmei; Xu, Kaijia; Wei, Xiaoxiao; Xu, Panli; Zhou, Yigang

    2017-11-01

    A novel magnetic extractant, PEG 4000 modified Fe 3 O 4 nanomaterial that coated with dianionic amino acid ionic liquid (Fe 3 O 4 @PEG@DAAAIL), was successfully synthesized and characterized. X-ray diffraction (XRD), transmission electron microscope (TEM), vibrating sample magnetometer (VSM), fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA) and zeta potentials were used to confirm that the novel nanocomposite was successfully synthesized. Subsequently, the prepared Fe 3 O 4 @PEG@DAAAIL nanocomposite was used as the extractant for trypsin coupled with magnetic solid-phase extraction (MSPE). The concentrations of trypsin in the supernatant were detected by UV-vis spectrophotometer at 278nm. The extraction ability turned out to be better than the other four kinds of extractants prepared in this work. Furthermore, the influence of a series of factors, such as extraction time and temperature, initial trypsin concentration, the value of pH and ionic strength, was systematically investigated. Under the optimal extraction condition, the extraction capacity for trypsin could reach up to 718.73mg/g, absolutely higher than that of other adsorbents reported. This satisfactory extraction capacity could be maintained unchangeable after at least eight days, and kept over 90% of initial extraction capacity after eight recycles. What's more, the activity of trypsin after extraction retained 92.29% of initial activity, verifying the biocompatibility of the prepared extractant. Finally, the developed Fe 3 O 4 @PEG@DAAAIL-MSPE method was successfully applied to the real sample analysis with satisfactory results. All of above proves the potential value of Fe 3 O 4 @PEG@DAAAIL-MSPE in the analysis of biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. ELISA analysis of soybean trypsin inhibitors in processed foods.

    Science.gov (United States)

    Brandon, D L; Bates, A H; Friedman, M

    1991-01-01

    Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

  19. Final characterization report for the 104-B-1 Tritium Vault and 104-B-2 Tritium Laboratory

    International Nuclear Information System (INIS)

    Encke, D.B.; Harris, R.A.

    1996-11-01

    This report is a compilation of the characterization data collected from the 104-B-1 Tritium Vault and the 104-B-2 Trillium Laboratory. The characterization activities were organized and implemented to evaluate the radiological status and identify any hazardous materials. The data contained in this report reflects the current conditions and status of the 104-B-1 Tritium Vault and 104-B-2 Tritium Laboratory. This information is intended to be utilized in support of future building decontamination and demolition, to allow for proper disposal of the demolition debris as required by the Washington Administrative Code, WAC 173-303, the Hanford Site Solid Waste Acceptance Criteria, WHC-EP-0063, and the Environmental Restoration Disposal Facility Waste Acceptance Criteria, BHI-00139. Based on the historical information and facility inspections, the only hazardous materials sampling and analysis activities necessary were to identify lead paint and asbestos containing materials (ACM) in the 104-B-1 Tritium Vault and the 104-B-2 Tritium Laboratory. Asbestos samples were obtained from the outer boundary of the roof areas to confirm the presence and type of asbestos containing fibers. Lead paint samples were obtained to confirm the presence and quantity of lead paint on the roof trim, doors and vents

  20. Bifenthrin activates homotypic aggregation in human T-cell lines.

    Science.gov (United States)

    Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

    2006-03-01

    Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

  1. Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Huimin; Bao, Huimin; Zhang, Luyan; Chen, Gang, E-mail: gangchen@fudan.edu.cn

    2014-10-03

    Highlights: • Trypsin was immobilized on miniature incandescent bulbs via chitosan coating. • The bulbs acted as enzymatic reactors and the generators of infrared radiation. • The bulb bioreactors were successfully employed in infrared-assisted proteolysis. • The proteolysis could accomplish within 5 min with high sequence coverages. - Abstract: A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL{sup −1} each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.

  2. Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery.

    Science.gov (United States)

    Chen, Yan; Ding, Jiawang; Qin, Wei

    2012-12-01

    A potentiometric biosensor for the determination of trypsin is described based on current-controlled reagent delivery. A polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation exchanger is used for in situ generation of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which the protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5UmL(-1) with a detection limit of 0.3UmL(-1). The proposed detection strategy provides a rapid and reagentless way for the detection of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Chemically modified, immobilized trypsin reactor with improved digestion efficiency

    NARCIS (Netherlands)

    Freije, J.R.; Mulder, P.P.; Werkman, W.; Rieux, L.; Niederlander, H.A G; Verpoorte, Sabeth; Bischoff, Rainer

    2005-01-01

    Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was

  4. 28 CFR 39.104-39.109 - [Reserved

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false [Reserved] 39.104-39.109 Section 39.104-39.109 Judicial Administration DEPARTMENT OF JUSTICE ENFORCEMENT OF NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES CONDUCTED BY THE DEPARTMENT OF JUSTICE §§ 39.104-39.109 [Reserved] ...

  5. 49 CFR 28.104-28.109 - [Reserved

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false [Reserved] 28.104-28.109 Section 28.104-28.109 Transportation Office of the Secretary of Transportation ENFORCEMENT OF NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES CONDUCTED BY THE DEPARTMENT OF TRANSPORTATION §§ 28.104-28.109 [Reserved] ...

  6. Study on transformation of cowpea trypsin inhibitor gene into ...

    African Journals Online (AJOL)

    Cowpea Trypsin Inhibitor (CpTI) gene was transferred into cauliflower by agrobacterium-mediated transformation method, and 14 transgenic cauliflower plants were obtained. Cotyledons and hypocotyls were used as explants. The putative transformants were assayed by PCR and Southern blotting analysis. The results ...

  7. Anti-Ma2 paraneoplastic encephalitis associated with testicular germ cell tumor treated by carboplatin, etoposide and bleomycin.

    Science.gov (United States)

    Kimura, Masaki; Onozawa, Mizuki; Fujisaki, Akira; Arakawa, Takashi; Takeda, Katsuhiko; Dalmau, Joseph; Hattori, Kazunori

    2008-10-01

    Anti-Ma2-associated encephalitis is a paraneoplastic disorder that predominantly affects the limbic system, diencephalon and brainstem, and is usually associated with tumors of the testis. We report a 35-year-old man with a right testicular mass who presented with multiple neurological complains, and clinical, serological and radiological features compatible with anti-Ma2-associated encephalitis. After three courses of carboplatin, etoposide and bleomycin for metastatic testicular germ-cell tumor, all elevated tumor markers normalized and the retroperitoneal metastases disappeared, but the neurological disorder deteriorated. To our knowledge, this is the first case in which orchiectomy followed by carboplatin, etoposide and bleomycin for a testicular tumor with anti-Ma2 encephalitis was performed.

  8. Cancer cells cause vascular endothelial cell (vEC) retraction via 12(S)HETE secretion; the possible role of cancer cell derived microparticle.

    Science.gov (United States)

    Uchide, Keiji; Sakon, Masato; Ariyoshi, Hideo; Nakamori, Syouji; Tokunaga, Masaru; Monden, Morito

    2007-02-01

    Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7. In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM. These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.

  9. Trypsin digest protocol to analyze the retinal vasculature of a mouse model.

    Science.gov (United States)

    Chou, Jonathan C; Rollins, Stuart D; Fawzi, Amani A

    2013-06-13

    Trypsin digest is the gold standard method to analyze the retinal vasculature (1-5). It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations (6,7). In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

  10. Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

    Science.gov (United States)

    Silva, Marciele S; Ribeiro, Suzanna Ff; Taveira, Gabriel B; Rodrigues, Rosana; Fernandes, Katia Vs; Carvalho, André O; Vasconcelos, Ilka Maria; Mello, Erica Oliveira; Gomes, Valdirene M

    2017-08-01

    During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P R Anil [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Varma, H K [Bioceramics Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Kumary, T V [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India)

    2007-03-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function.

  12. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Kumar, P R Anil; Varma, H K; Kumary, T V

    2007-01-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function

  13. Atomic-scale investigation of the interactions between tetrabromobisphenol A, tetrabromobisphenol S and bovine trypsin by spectroscopies and molecular dynamics simulations

    International Nuclear Information System (INIS)

    Ding, Keke; Zhang, Huanxin; Wang, Haifei; Lv, Xuan; Pan, Liumeng; Zhang, Wenjing; Zhuang, Shulin

    2015-01-01

    Highlights: • The interaction of TBBPA/TBBPS with bovine trypsin was deciphered for the first time. • The fluorescence of bovine trypsin was quenched in a concentration-dependent mode. • TBBPA and TBBPS bind at the ANS binding site with distinct binding modes. • TBBPS has a higher binding affinity toward bovine trypsin than TBBPA. • Our in vitro and in silico approach is helpful to assess risk of TBBPA-related BFRs. - Abstract: Tetrabromobisphenol A (TBBPA) and its replacement alternative tetrabromobisphenol S (TBBPS) are used widely as brominated flame retardants (BFRs). However, the potential risk of their effects on bovine trypsin remains largely unknown. We investigated the effects of TBBPA and TBBPS to bovine trypsin by the fluorescence spectroscopy, circular dichroism and molecular dynamics (MD) simulations. They statically quenched the intrinsic fluorescence of bovine trypsin in a concentration-dependent mode and caused slight red-shifted fluorescence. The short and long fluorescence lifetime decay components of bovine trypsin were both affected, partly due to the disturbed microenvironmental changes of Trp215. The β-sheet content of bovine trypsin was significantly reduced from 82.4% to 75.7% and 76.6% by TBBPA and TBBPS, respectively, possibly impairing the physiological function of bovine trypsin. TBBPA and TBBPS bind at the 8-anilinonaphthalene-1-sulfonate (ANS) binding site with an association constant of 1.09 × 10 4 M −1 and 2.41 × 10 4 M −1 at 298 K, respectively. MD simulations revealed that van der Waals interactions and hydrogen bond interactions are dominant for TBBPA, whereas electrostatic interactions are critical for TBBPS. Our in vitro and in silico studies are beneficial to the understanding of risk assessment and future design of environmental benign BFRs.

  14. Rapid internalization of the insulin receptor in rat hepatoma cells

    International Nuclear Information System (INIS)

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-01-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

  15. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  16. Early (pre–8 Ma) fault activity and temporal strain accumulation in the central Indian Ocean

    Digital Repository Service at National Institute of Oceanography (India)

    Krishna, K.S.; Bull, J.M.; Scrutton, R.A.

    -reflection profiles within the central Indian Ocean demonstrate that compressional activity started much earlier than previously thought, at around 15.4-13.9 Ma. From reconstructions of fault activity histories, it is shown that 12% of the total reverse fault...

  17. Effect of traditional processing methods on the β-carotene, ascorbic acid and trypsin inhibitor content of orange-fleshed sweet potato for production of amala in Nigeria.

    Science.gov (United States)

    Yusuf, Abbas Bazata; Fuchs, Richard; Nicolaides, Linda

    2016-05-01

    The aim of the work was to study the effect of traditional processing methods on the β-carotene, ascorbic acid and trypsin inhibitor contents of orange-fleshed sweet potato amala. The most common sweet potato in Nigeria is white or yellow fleshed, which is very low in provitamin A. However, efforts are underway to promote orange-fleshed sweet potato to improve provitamin A intake. This paper describes how orange-fleshed sweet potato slices were traditionally processed into amala, which is increasingly consumed in Nigeria. The study revealed that both the cold and hot fermentation methods resulted in increased vitamin A levels and lower vitamin C levels in orange-fleshed sweet potato. Further processing to make amala resulted in a fall in both vitamin A and C content. The study found an increase in trypsin inhibitor activity following the cold-water fermentation and a decrease following the hot-water fermentation compared to raw orange-fleshed sweet potato. Trypsin inhibitor activity in amala produced using both the cold and hot methods was below detectable levels. The results indicate that amala produced from traditionally fermented orange-fleshed sweet potato could be a good source of vitamins A and C for the rural poor and that the processing removes any potential negative effects of trypsin inhibitors. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  18. An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).

    Science.gov (United States)

    Conners, Rebecca; Konarev, Alexander V; Forsyth, Jane; Lovegrove, Alison; Marsh, Justin; Joseph-Horne, Timothy; Shewry, Peter; Brady, R Leo

    2007-09-21

    The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K(i) and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.

  19. Effect of radiation and soaking on trypsin inhibitor and protein content of chickpea (Cicer arietinum L.)

    International Nuclear Information System (INIS)

    Sattar, A.; Atta, S.; Akhtar, M.A.

    1990-01-01

    Composition of Ascochyta-blight resistant chickpea for proximate components, vitamins, energy and trypsin inhibitor, was determined. The influence of irradiation and soaking at ambient temperatures (25-35deg C) on trypsin inhibitor activity (TIA) and protein content of chickpea, was investigated. A significant linear relation (r = -0.960 to -0.987) was found between the loss of TIA and soaking time of irradiated and unirradiated seeds (p < 0.05) and the rate of loss increased with increasing radiation dose (0.25-1.00 kGy). However, effect of radiation alone was negligible. Maximum decrease (30.7%) in TIA (from 330.0 to 228.6 TiU/g) occurred during soaking for 12 h of 1.00 kGy sample. The protein contents increased from an initial value of 21.7% to 23.4% and 22.7% as a result of soaking for 12 h in tap and distilled waters, respectively. Radiation treatment exhibited little or no effect. (author)

  20. [Ma2 antibody and multiple mononeuropathies].

    Science.gov (United States)

    Ayrignac, X; Castelnovo, G; Landrault, E; Fayolle, H; Pers, Y-M; Honnorat, J; Campello, C; Figarella-Branger, D; Labauge, P

    2008-01-01

    Anti-Ma2 antibodies belong to a family of onconeuronal antibodies that target proteins expressed in brain, testis and several tumors. Previously observed in patients presenting with limbic encephalitis, they seem to be associated with several other paraneoplastic syndromes. We report the case of a 73-year-old woman presenting sensory and motor neuropathy associated with non-small-cell lung cancer who had Ma2-antibodies.

  1. Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

    Science.gov (United States)

    Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd; Baig, Mohd Affan; Qureshi, M Irfan; Ali, Sher; Fatima, Sadaf

    2016-01-01

    This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI

  2. Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

    Directory of Open Access Journals (Sweden)

    Tooba Naz Shamsi

    Full Text Available This study was aimed to purify and characterize the Protease inhibitor (PI from a plant Allium sativum (garlic with strong medicinal properties and to explore its phytodrug potentials.Allium sativum Protease Inhibitor (ASPI was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD spectroscopy.ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM. The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites

  3. Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis

    International Nuclear Information System (INIS)

    Jones, Steven; Boisvert, Annie; Naghi, Andrada; Hullin-Matsuda, Françoise; Greimel, Peter; Kobayashi, Toshihide; Papadopoulos, Vassilios; Culty, Martine

    2016-01-01

    Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10 μM GEN + 10 μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN + MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10 μM combined GEN + MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN + MEHP. We propose a working model for GEN + MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally

  4. Episodic nature of continental arc activity since 750 Ma: A global compilation

    Science.gov (United States)

    Cao, Wenrong; Lee, Cin-Ty A.; Lackey, Jade Star

    2017-03-01

    Continental arcs have been recently hypothesized to outflux large amounts of CO2 compared to island arcs so that global flare-ups in continental arc magmatism might drive long-term greenhouse events. Quantitative testing of this hypothesis, however, has been limited by the lack of detailed studies on the spatial distribution of continental arcs through time. Here, we compile a worldwide database of geological maps and associated literature to delineate the surface exposure of granitoid plutons, allowing reconstruction of how the surface area addition rate of granitoids and the length of continental arcs have varied since 750 Ma. These results were integrated into an ArcGIS framework and plate reconstruction models. We find that the spatial extent of continental arcs is episodic with time and broadly matches the detrital zircon age record. Most vigorous arc magmatism occurred during the 670-480 Ma and the 250-50 Ma when major greenhouse events are recognized. Low continental arc activity characterized most of the Cryogenian, middle-late Paleozoic, and Cenozoic when climate was cold. Our results indicate that plate tectonics is not steady, with fluctuations in the nature of subduction zones possibly related in time to the assembly and dispersal of continents. Our results corroborate the hypothesis that variations in continental arc activity may play a first order role in driving long-term climate change. The dataset presented here provides a quantitative basis for upscaling continental arc processes to explore their effects on mountain building, climate, and crustal growth on a global scale.

  5. Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications.

    Science.gov (United States)

    Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Xiaoyun

    2014-09-23

    Pre-fractionation by gel electrophoresis is often combined with liquid chromatography-mass spectrometry (LC-MS) for large-scale profiling of complex protein samples. An essential component of this widely applied proteomic platform is in-gel protein digestion. In nearly two decades of practicing this approach, an extremely high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. Here we report that trypsin autolysis products contribute to the bulk of chemical noise in in-gel digestion and remarkably we found evidence that the amount of trypsin can be slashed by an order of magnitude with comparable digestion performance. By revising perhaps the most critical element of this decade-old digestion protocol, the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered cost due to enzyme expenditure. More importantly, substantially reduced chemical noise (i.e., trypsin self-cleavage products) as a result of less enzyme usage translates into more protein identifications when limited amounts of samples are the interest of interrogation. In-gel digestion is one of the most widely used methods in proteomics. An exceedingly high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. This requirement has been faithfully kept in nearly two decades of practicing this approach. Here we report that trypsin concentration can be slashed by at least an order of magnitude while still providing comparable digestion performance. Thus the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered enzyme cost. More importantly, substantially reduced chemical noise (i.e., trypsin autolysis products) due to less enzyme usage translates into ~30% more protein identifications when limited amounts of protein samples are analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. New analogues of Cucurbita maxima trypsin inhibitor III (CMTI III) with simplified structure.

    Science.gov (United States)

    Rolka, K; Kupryszewski, G; Rózycki, J; Ragnarsson, U; Zbyryt, T; Otlewski, J

    1992-10-01

    Seven new analogues of trypsin inhibitor CMTI III were obtained by solid-phase peptide synthesis. Three analogues contained only two, instead of three, disulfide bridges, whereas the molecules of the next four analogues were shortened at the N- and/or C-terminus. The elimination of one disulfide bridge in CMTI III induces a decrease in the association equilibrium constants by 6-7 orders of magnitude, whereas the removal of one, two or three amino-acid residues at the N- and/or C-terminus does not significantly affect the activity.

  7. Anti-Ma and anti-Ma2-associated paraneoplastic neurological syndromes.

    Science.gov (United States)

    Ortega Suero, G; Sola-Valls, N; Escudero, D; Saiz, A; Graus, F

    Analyse the clinical profile, associated tumour types, and response to treatment of paraneoplastic neurological syndromes associated with antibodies against Ma proteins. A retrospective study of patients with antibodies against Ma proteins identified in a neuroimmunology laboratory of reference. Of the 32 patients identified, 20 showed reactivity against Ma2 only (anti-Ma2 antibodies), 11 against Ma1 and Ma2 (anti-Ma antibodies), and 1 with reactivity against Ma1 only (anti-Ma1 antibodies). The most common clinical presentations were limbic encephalopathy, diencephalic dysfunction, or brainstem encephalopathy, frequently appearing as a combination of these features. Three patients had isolated cerebellar dysfunction with anti-Ma antibodies, and 2 exhibited peripheral nervous system syndrome with anti-Ma2 antibodies. Testicular tumours were the most common neoplasms (40%) in the anti-Ma2 cases. In the group associated with anti-Ma1 antibodies, the most common were lung tumours (36%), followed by testicular tumours. All idiopathic cases were reactive to Ma2. The clinical outcome was significantly better in the anti-Ma2 group. The patient with anti-Ma1 presented with limbic encephalitis and brainstem dysfunction associated with lymphoepithelioma of the bladder. Specifically determining the different reactivities of anti-Ma protein antibodies in order to differentiate between Ma1 and Ma2 antibodies is important because anti-Ma2-associated paraneoplastic syndromes have a better outcome. Lastly, this study is the first to confirm that there may be cases that react exclusively to antibodies against Ma1. Copyright © 2016 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  8. Trypsin level in gallbladder bile and ductitis and width of the cystic duct.

    Science.gov (United States)

    Vracko, J; Wiechel, K L

    2000-01-01

    The change from laparotomy to laparoscopy for cholecystectomy has raised the question of how to manage concomitant bile duct stones. The present-day interest--and controversy--has focused on a transcystic approach reported to be feasible in 66-96% of cases, but without explaining the necessary prerequisite: the widening of the cystic duct. The cystic duct, wide mainly in patients with bile duct stones, has been reported to be highly variable: from strictured to very wide. The present study aims at comparing the trypsin level in the gallbladder bile and the cystic duct morphology and width in patients with and without bile duct stones. A prospective series of 63 gallstone patients, 30 with and 33 without bile duct stones (controls), underwent cholecystectomy and bile duct clearance. The study includes the trypsin level in the gallbladder bile, the width and morphology of the cystic duct, and the size of the gallstones. The patients with bile duct stones had, in contrast to the controls, higher trypsin levels in the gallbladder bile (P extraction feasible.

  9. Proliferation of Schwann cells induced by axolemmal and myelin membranes

    International Nuclear Information System (INIS)

    Dinneen, M.

    1985-01-01

    Purified Schwann Cells were cultured from neonatal rat sciatic nerve using a modification of the method of Brockes. Schwann cells and contaminating fibroblasts were unambiguously identified using fluorescent antibodies of 2'3' cyclic nucleotide 3'-phosphodiesterase and the thy 1.1 antigen respectively. The Schwann cells were quiescent unless challenged with mitogens. They proliferated rapidly in response to the soluble mitogen, cholera toxin, or to membrane fractions from rat CNS or PNS, prepared by the method of DeVries. Mitogenic activity was present in both axolemmal and myelin enriched fractions and promoted a 10-15 fold increase in the rate of 3 H-thymidine uptake. The axolemmal mitogen was sensitive to heat (80 0 C for 10 minutes), trypsin digestion (0.05% x 30 mins) or to treatment with endoglycosidase D, suggesting that it could be a glycoprotein. Fifty percent of the axolemmal mitogenic activity was solubilized in 1% octyl-glucoside. The solubilized material, however, was very unstable and further purification was not possible. The myelin associated mitogenic activity was markedly different. It was resistant to freeze thaw cycles, trypsin digestion of endoglycosidase treatment and the activity was actually enhanced by heating at 100 0 C for two hours. It is proposed that the axolemmal activity is responsible for Schwann cell proliferation during development and that the myelin associated activity promotes Schwann cell proliferation during Wallerian degeneration

  10. Detection of five tumor markers in lung cancer by trypsin digestion of sputum method

    International Nuclear Information System (INIS)

    Lin Min; Nong Tianlei; Liu Daying

    2011-01-01

    To explore the detection of five tumor markers by trypsin digestion of sputum in the diagnosis of lung cancer, the samples of sputum in patients with lung cancer and benign lung disease were digested by trypsin and used to measure five tumor markers. The results showed that the sputum were well digested by 6% trypsin at pH8 and no affect on the determination of tumor markers. The CEA, CA125, CA153, CA211 and NSE levels in lung cancer group were significantly higher than that of in benign group (P<0.05). The sputum CEA and CA125 levels were significantly higher than that of the serum levels (P<0.05). The detection of sputum CEA, CA125, CA153, CA211 and NSE levels have clinical value in the diagnosis of lung cancer. When combined with other diagnostic methods,it might be helpful for further diagnosis in non confirmed lung cancer patients. (authors)

  11. [In-line leukocyte depletion ov thrombocytapheresis concentrates with the Fresenius-AS-104 cell separator].

    Science.gov (United States)

    Zeiler, T; Kretschmer, V

    1997-01-01

    This study reports on in-line filtration of 72 platelet concentrates (PC) collected by the Fresenius AS 104 cell separator, using the new C4F sets with integrated leukocyte filters (Biofil P plus). 72 volunteer donors, automatic counts of platelets, microscopical counting of residual leukocytes with the Nageotte chamber, GMP-140 by flow cytometrie, beta-thromboglobulin release, platelet aggregation (ADP, collagen). Filtration reduced leukocytes by 98.5%. Residual leukocyte contamination remained clearly below 5 x 10(6) (mean 0.5 +/- 0.6 x 10(6), maximum 2.8 x 10(6). Platelet loss by filtration was found to be between 27.4 and 0.7% (median 8.5%). Filtration caused a significant decrease of platelet aggregability (p < 0.005), but no significant increase of beta-thromboglobulin release and only a slight decrease of GMP-140 expression. From these data can be concluded that in-line filtration was highly efficient with acceptable platelet retention. No significant platelet activation could be observed in the PC. The decrease of platelet aggregability have been due to the reduction of activated platelets which are believed to show reduced in vivo survival.

  12. Orchiectomy for suspected microscopic tumor in patients with anti-Ma2-associated encephalitis.

    Science.gov (United States)

    Mathew, R M; Vandenberghe, R; Garcia-Merino, A; Yamamoto, T; Landolfi, J C; Rosenfeld, M R; Rossi, J E; Thiessen, B; Dropcho, E J; Dalmau, J

    2007-03-20

    To report the presence of microscopic neoplasms of the testis in men with anti-Ma2-associated encephalitis (Ma2-encephalitis) and to discuss the clinical implications. Orchiectomy specimens were examined using immunohistochemistry with Ma2 and Oct4 antibodies. Among 25 patients with Ma2-encephalitis younger than 50 years, 19 had germ-cell tumors, and 6 had no evidence of cancer. These 6 patients underwent orchiectomy because they fulfilled five criteria: 1) demonstration of anti-Ma2 antibodies in association with MRI or clinical features compatible with Ma2-encephalitis, 2) life-threatening or progressive neurologic deficits, 3) age Ma2 was expressed by neoplastic cells in three of three patients examined. Even though most patients had severe neurologic deficits at the time of orchiectomy (median progression of symptoms, 10 months), 4 had partial improvement and prolonged stabilization (8 to 84 months, median 22.5 months) and two did not improve after the procedure. In young men with Ma2-encephalitis, 1) the disorder should be attributed to a germ-cell neoplasm of the testis unless another Ma2-expressing tumor is found, 2) negative tumor markers, ultrasound, body CT, or PET do not exclude an intratubular germ-cell neoplasm of the testis, and 3) if no tumor is found, the presence of the five indicated criteria should prompt consideration of orchiectomy.

  13. Molecular and clinical diversity in paraneoplastic immunity to Ma proteins.

    Science.gov (United States)

    Rosenfeld, M R; Eichen, J G; Wade, D F; Posner, J B; Dalmau, J

    2001-09-01

    Antibodies to Ma1 and Ma2 proteins identify a paraneoplastic disorder that affects the limbic system, brain stem, and cerebellum. Preliminary studies suggested the existence of other Ma proteins and different patterns of immune response associated with distinct neurologic symptoms and cancers. In this study, our aim was to isolate the full-length sequence of Ma2 and new family members, identify the major autoantigen of the disorder, and extend the dinical-immunological analysis to 29 patients. Sera from selected patients were used to probe a brainstem cDNA library and isolate the entire Ma2 gene and a new family member, Ma3. Ma3 mRNA is ubiquitously expressed in brain, testis, and several systemic tissues. The variable cellular expression of Ma proteins and analysis of protein motifs suggest that these proteins play roles in the biogenesis of mRNA. Immunoblot studies identify Ma2 as the major autoantigen with unique epitopes recognized by all patients' sera. Eighteen patients had antibodies limited to Ma2: they developed limbic, hypothalamic, and brainstem encephalitis, and 78% had germ-cell tumors of the testis. Eleven patients had antibodies to Ma2 and additional antibodies to Ma1 and/or Ma3; they usually developed additional cerebellar symptoms and more intense brainstem dysfunction, and 82% of these patients had tumors other than germ-cell neoplasms. Overall, 17 of 24 patients (71%) with brain magnetic resonance imaging studies had abnormalities within or outside the temporal lobes, some as contrast-enhancing nodular lesions. A remarkable finding of immunity to Ma proteins is that neurologic symptoms may improve or resolve. This improvement segregated to a group of patients with antibodies limited to Ma2.

  14. A prospective randomized trial of Kotase ® (Bromelain + Trypsin) in ...

    African Journals Online (AJOL)

    International Journal of Medicine and Health Development. Journal Home · ABOUT THIS ... A prospective randomized trial of Kotase® (Bromelain + Trypsin) in the management of post-operative abdominal wounds at the University of Nigeria Teaching Hospital Enugu, Nigeria. Emmanuel R Ezeome, Aloy E Aghaji ...

  15. Influence of carbohydrates on the interaction of procyanidin B3 with trypsin.

    Science.gov (United States)

    Gonçalves, Rui; Mateus, Nuno; De Freitas, Victor

    2011-11-09

    The biological properties of procyanidins, in particular their inhibition of digestive enzymes, have received much attention in the past few years. Dietary carbohydrates are an environmental factor that is known to affect the interaction of procyanidins with proteins. This work aimed at understanding the effect of ionic food carbohydrates (polygalacturonic acid, arabic gum, pectin, and xanthan gum) on the interaction between procyanidins and trypsin. Physical-chemical techniques such as saturation transfer difference-NMR (STD-NMR) spectroscopy, fluorescence quenching, and nephelometry were used to evaluate the interaction process. Using STD-NMR, it was possible to identify the binding of procyanidin B3 to trypsin. The tested carbohydrates prevented the association of procyanidin B3 and trypsin by a competition mechanism in which the ionic character of carbohydrates and their ability to encapsulate procyanidins seem crucial leading to a reduction in STD signal and light scattering and to a recovery of the proteins intrinsic fluorescence. On the basis of these results, it was possible to grade the carbohydrates in their aggregation inhibition ability: XG > PA > AG ≫ PC. These effects may be relevant since the coingestion of procyanidins and ionic carbohydrates are frequent and furthermore since these might negatively affect the antinutritional properties ascribed to procyanidins in the past.

  16. Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

    Science.gov (United States)

    Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

    2015-09-01

    The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

  17. Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

    Science.gov (United States)

    de P G Gomes, Angélica; Dias, Simoni C; Bloch, Carlos; Melo, Francislete R; Furtado, José R; Monnerat, Rose G; Grossi-de-Sá, Maria F; Franco, Octávio L

    2005-02-01

    Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

  18. A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

    Science.gov (United States)

    Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

    2016-03-01

    The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Number of nuclei, mitotic activity and cell length in Cladophora sp thallus treated with cadmium and chromium

    Directory of Open Access Journals (Sweden)

    Monika Krajewska

    2014-01-01

    Full Text Available Cladophora sp., a fresh water, filamentous, multi-nucleate alga growing 16 days in the presence of cadmium and chromium at concentrations 10-4 10-8M was the subject of the experiment. Chromium ions reduced the number of nuclei and mitotic activity, and disturbed the correlation between cell length and number of nuclei, more than cadmium ions. Moreover, both tested metals caused the disappearance of cells with numerous nuclei with time of the culture. Only during the first (1-4 days of culture for both metals the concentration of 10-4M and especially of 10-8M increased the number of nuclei, mitotic index and the length of cells. Apical cells were more sensitive to metals than other thallus cells.

  20. Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

    Directory of Open Access Journals (Sweden)

    Andrade A.Q.

    2002-01-01

    Full Text Available Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6 mesangial cells (MC and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05 in the cell lysate (43.5 ± 5.7 ng h-1 10(6 cells than in the culture medium (12.5 ± 2.5 ng h-1 10(6 cells. Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 ± 3.1 and 3.9 ± 0.9 ng h-1 10(6 cells. Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

  1. Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

    Czech Academy of Sciences Publication Activity Database

    Štosová, T.; Šebela, M.; Řehulka, Pavel; Šedo, O.; Havliš, J.; Zdráhal, Z.

    2008-01-01

    Roč. 376, č. 1 (2008), s. 94-102 ISSN 0003-2697 Institutional research plan: CEZ:AV0Z40310501 Keywords : MALDI-TOF MS * Streptomyces griseus * trypsin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.088, year: 2008

  2. Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

    Science.gov (United States)

    Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

    2017-08-30

    Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

  3. The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients.

    Science.gov (United States)

    Hoang, Van T; Buss, Eike C; Wang, Wenwen; Hoffmann, Isabel; Raffel, Simon; Zepeda-Moreno, Abraham; Baran, Natalia; Wuchter, Patrick; Eckstein, Volker; Trumpp, Andreas; Jauch, Anna; Ho, Anthony D; Lutz, Christoph

    2015-08-01

    To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible. © 2014 UICC.

  4. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

    International Nuclear Information System (INIS)

    Vega, Sebastián L.; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L.; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V.

    2017-01-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging-derived” parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. - Highlights: • High-content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages. • Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors. • A new class of cancer-mitigating biomaterials was identified based on image

  5. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

    Energy Technology Data Exchange (ETDEWEB)

    Vega, Sebastián L. [Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ (United States); Liu, Er; Arvind, Varun [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ (United States); Bushman, Jared [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); School of Pharmacy, University of Wyoming, Laramie, WY (United States); Sung, Hak-Joon [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Becker, Matthew L. [Department of Polymer Science and Engineering, University of Akron, Akron, OH (United States); Lelièvre, Sophie [Department of Basic Medical Sciences, Purdue University, West Lafayette, IN (United States); Kohn, Joachim [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); Vidi, Pierre-Alexandre, E-mail: pvidi@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC (United States); Moghe, Prabhas V., E-mail: moghe@rutgers.edu [Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ (United States); Department of Biomedical Engineering, Rutgers University, Piscataway, NJ (United States)

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging-derived” parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. - Highlights: • High-content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages. • Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors. • A new class of cancer-mitigating biomaterials was identified based on image

  6. [Anti-Ma2-associated encephalitis and paraneoplastic limbic encephalitis].

    Science.gov (United States)

    Yamamoto, Tomotaka; Tsuji, Shoji

    2010-08-01

    proportion of the patients, approximately one-third show neurological improvement and another 20 - 40% stabilize in response to treatment, including immunotherapy and/or tumor treatment. Patients who have limited CNS involvement and testicular tumors with complete response to therapy are more likely to show neurological improvement. This fact emphasizes the importance of early diagnosis and prompt initiation of therapy. However, it should be noted that even carcinoma in situ, which is difficult to detect can cause severe neurological disorders. In this respect, it is useful to highlight that anti-Ma2 encephalitis is almost always associated with testicular germ cell tumors in men younger than 50 years. We experienced a 40-year-old patient with severe hypokinesis caused by anti-Ma2 encephalitis associated with bilateral intratubular germ-cell neoplasm of the testes. In older men and women, non-small-cell lung cancer is most common but various types of cancers are reported to be associated. In this study,in addition to reviewing the above case we have reviewed the significance of anti-Ma2 antibodies in the diagnosis of anti-Ma2 encephalitis and the clinical features of this disease.

  7. Magnetically modified bacterial cellulose: A promising carrier for immobilization of affinity ligands, enzymes, and cells

    Energy Technology Data Exchange (ETDEWEB)

    Baldikova, Eva [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Pospiskova, Kristyna [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Ladakis, Dimitrios; Kookos, Ioannis K. [Department of Chemical Engineering, University of Patras, 26504 Patras, Rio (Greece); Koutinas, Apostolis A. [Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, Athens 11855 (Greece); Safarikova, Mirka [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Safarik, Ivo, E-mail: safarik@nh.cas.cz [Global Change Research Institute, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic); Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2017-02-01

    Bacterial cellulose (BC) produced by Komagataeibacter sucrofermentans was magnetically modified using perchloric acid stabilized magnetic fluid. Magnetic bacterial cellulose (MBC) was used as a carrier for the immobilization of affinity ligands, enzymes and cells. MBC with immobilized reactive copper phthalocyanine dye was an efficient adsorbent for crystal violet removal; the maximum adsorption capacity was 388 mg/g. Kinetic and thermodynamic parameters were also determined. Model biocatalysts, namely bovine pancreas trypsin and Saccharomyces cerevisiae cells were immobilized on MBC using several strategies including adsorption with subsequent cross-linking with glutaraldehyde and covalent binding on previously activated MBC using sodium periodate or 1,4-butanediol diglycidyl ether. Immobilized yeast cells retained approximately 90% of their initial activity after 6 repeated cycles of sucrose solution hydrolysis. Trypsin covalently bound after MBC periodate activation was very stable during operational stability testing; it could be repeatedly used for ten cycles of low molecular weight substrate hydrolysis without loss of its initial activity. - Highlights: • Bacterial cellulose was magnetically modified with magnetic fluid. • Magnetic cellulose is an efficient carrier for affinity ligands. • Enzymes and cells can be efficiently immobilized to magnetic cellulose.

  8. The Banana Fruit SINA Ubiquitin Ligase MaSINA1 Regulates the Stability of MaICE1 to be Negatively Involved in Cold Stress Response.

    Science.gov (United States)

    Fan, Zhong-Qi; Chen, Jian-Ye; Kuang, Jian-Fei; Lu, Wang-Jin; Shan, Wei

    2017-01-01

    The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis . Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi- in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

  9. Biological Activity Alterations of Human Amniotic Membrane Pre and Post Irradiation Tissue Banking.

    Science.gov (United States)

    Nemr, Waleed; Bashandy, A S; Araby, Eman; Khamiss, O

    Innate immunity of Human Amniotic Membrane (HAM) and its highly active secretome that rich with various types of growth factors and anti-inflammatory substances proposed it as a promising material for many medical studies and applications. This study evaluate the biological activity of cultivated HAM pre and post tissue banking process in which freeze-dried HAM was sterilized by 25 KGray (kGy) dose of γ radiation. The HAM's antimicrobial activity, viability, growth of isolated human amniotic epithelial cells (HAECs), hematopoietic stimulation of co-cultivated murine bone marrow cells (mammalian model), scaffold efficiency for fish brain building up (non-mammalian model) and self re-epithelialization after trypsin denuding treatment were examined as supposed biological activity features. Native HAM revealed viability indications and was active to kill all tested microorganisms; 6 bacterial species (3 Gram-positive and 3 Gram-negative) and Candida albicans as a pathogenic fungus. Also, HAM activity promoted colony formation of murine hematopoietic cells, Tilapia nilotica brain fragment building-up and self re-epithelialization after trypsin treatment. In contrary, radiation-based tissue banking of HAM caused HAM cellular death and consequently lacked almost all of examined biological activity features. Viable HAM was featured with biological activity than fixed HAM prepared by irradiation tissue banking.

  10. 12 CFR 410.104-410.109 - [Reserved

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 4 2010-01-01 2010-01-01 false [Reserved] 410.104-410.109 Section 410.104-410.109 Banks and Banking EXPORT-IMPORT BANK OF THE UNITED STATES ENFORCEMENT OF NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES CONDUCTED BY EXPORT-IMPORT BANK OF THE UNITED STATES §§ 410...

  11. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

    Science.gov (United States)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-01

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

  12. Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

    Science.gov (United States)

    Dubey, J P; Sundar, N; Kwok, O C H; Saville, W J A

    2013-09-01

    The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals. Published by Elsevier B.V.

  13. 34 CFR 104.33 - Free appropriate public education.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Free appropriate public education. 104.33 Section 104.33 Education Regulations of the Offices of the Department of Education OFFICE FOR CIVIL RIGHTS, DEPARTMENT OF EDUCATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING...

  14. 33 CFR 80.135 - Hull, MA to Race Point, MA.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Hull, MA to Race Point, MA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Atlantic Coast § 80.135 Hull, MA to Race Point, MA. (a... the east coast of Massachusetts from the easternmost radio tower at Hull, charted in approximate...

  15. Solar Cell Polymer Based Active Ingredients PPV and PCBM

    Science.gov (United States)

    Hardeli, H.; Sanjaya, H.; Resikarnila, R.; Nitami H, R.

    2018-04-01

    A polymer solar cell is a solar cell based on a polymer bulk heterojunction structure using the method of thin film, which can convert solar energy into electrical energy. Absorption of light is carried by active material layer PPV: PCBM. This study aims to make solar cells tandem and know the value of converting solar energy into electrical energy and increase the value of efficiency generated through morphological control, ie annealing temperature and the ratio of active layer mixture. The active layer is positioned above the PEDOT:PSS layer on ITO glass substrate. The characterization results show the surface morphology of the PPV:PCBM active layer is quite evenly at annealing temperature of 165 ° C. The result of conversion of electrical energy with a UV light source in annealing samples with temperature 165 ° C is 0.03 mA and voltage of 4.085 V with an efficiency of 2.61% and mixed ratio variation was obtained in comparison of P3HT: PCBM is 1: 3

  16. A phase I trial of PR-104, a pre-prodrug of the bioreductive prodrug PR-104A, given weekly to solid tumour patients

    International Nuclear Information System (INIS)

    McKeage, Mark J; Gu, Yongchuan; Wilson, William R; Hill, Andrew; Amies, Karen; Melink, Teresa J; Jameson, Michael B

    2011-01-01

    The phosphate ester PR-104 is rapidly converted in vivo to the alcohol PR-104A, a nitrogen mustard prodrug that is metabolised to hydroxylamine (PR-104H) and amine (PR-104M) DNA crosslinking agents by one-electron reductases in hypoxic cells and by aldo-keto reductase 1C3 independently of oxygen. In a previous phase I study using a q 3 week schedule of PR-104, the maximum tolerated dose (MTD) was 1100 mg/m 2 and fatigue, neutropenic fever and infection were dose-limiting. The primary objective of the current study was to determine the dose-limiting toxicity (DLT) and MTD of weekly PR-104. Patients with advanced solid tumours received PR-104 as a 1-hour intravenous infusion on days 1, 8 and 15 every 28 days with assessment of pharmacokinetics on cycle 1 day 1. Twenty-six patients (pts) were enrolled (16 male/10 female; median age 58 yrs, range 30 to 70 yrs) who had received a median of two prior chemotherapy regimens (range, 0 to 3) for melanoma (8 pts), colorectal or anal cancer (3 pts), NSCLC (3 pts), sarcoma (3 pts), glioblastoma (2 pts), salivary gland tumours (2 pts) or other solid tumours (5 pts). PR-104 was administered at 135 mg/m 2 (3 pts), 270 mg/m 2 (6 pts), 540 mg/m 2 (6 pts), 675 mg/m 2 (7 pts) and 900 mg/m 2 (4 pts) for a median of two treatment cycles (range, 1 to 7 cycles) and five infusions (range, 1 to 18) per patient. Dose-limiting toxicities (DLTs) during cycle one included grade four thrombocytopenia at 540 mg/m 2 (1 of 6 pts) and grade four thrombocytopenia and neutropenia at 900 mg/m 2 (2 of 4 pts). At an intermediate dose of 675 mg/m 2 , there were no DLTs among a total of seven patients given 12 treatment cycles but all experienced moderate to severe (grade 2 to 4) haematological toxicity. Thrombocytopenia was delayed in its onset and nadir, and its recovery was protracted and incomplete in many patients. There were no complete or partial tumour responses. PR-104-induced thrombocytopenia and neutropenia correlated with plasma AUC of PR-104

  17. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    International Nuclear Information System (INIS)

    Highsmith, R.F.; Gallaher, M.J.

    1986-01-01

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  18. A capillary monolithic trypsin reactor for efficient protein digestion in online and offline coupling to ESI and MALDI mass spectrometry.

    Science.gov (United States)

    Spross, Jens; Sinz, Andrea

    2010-02-15

    We describe the preparation of a capillary trypsin immobilized monolithic enzyme reactor (IMER) for a rapid and efficient digestion of proteins down to the femtomole level. Trypsin was immobilized on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith using the glutaraldehyde technique. Digestion efficiencies of the IMER were evaluated using model proteins and protein mixtures as well as chemically cross-linked lysozyme regarding the addition of denaturants and increasing digestion temperature. The trypsin IMER described herein is applicable for the digestion of protein mixtures. Even at a 1000-fold molar excess of one protein, low-abundance proteins are readily identified, in combination with MS/MS analysis. An online setup of the IMER with reversed phase nano-HPLC separation and nano-ESI-MS/MS analysis was established. The great potential of the trypsin IMER for proteomics applications comprise short digestion times in the range of seconds to minutes, in addition to improved digestion efficiencies, compared to in-solution digestion.

  19. 34 CFR 104.38 - Preschool and adult education.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Preschool and adult education. 104.38 Section 104.38 Education Regulations of the Offices of the Department of Education OFFICE FOR CIVIL RIGHTS, DEPARTMENT OF EDUCATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL...

  20. 42 CFR 495.202 - Identification of qualifying MA organizations, MA-EPs and MA-affiliated eligible hospitals.

    Science.gov (United States)

    2010-10-01

    ... qualifying MA-affiliated eligible hospitals under the MA EHR incentive program are required to identify...-EPs and MA-affiliated eligible hospitals. 495.202 Section 495.202 Public Health CENTERS FOR MEDICARE... STANDARDS FOR THE ELECTRONIC HEALTH RECORD TECHNOLOGY INCENTIVE PROGRAM Requirements Specific to Medicare...

  1. Syringic acid from Tamarix aucheriana possesses antimitogenic and chemo-sensitizing activities in human colorectal cancer cells.

    Science.gov (United States)

    Abaza, Mohamed-Salah; Al-Attiyah, Raja'a; Bhardwaj, Radhika; Abbadi, Ghaneim; Koyippally, Mathew; Afzal, Mohammad

    2013-09-01

    For its variety of biological activities, Tamarix aucheriana (Decne.) Baum. (Tamaricaceae) has an extensive history as a traditional Arab medicine. Antimitogenic and chemo-sensitizing activities of syringic acid (SA) were studied against human colorectal cancer. Chromatographic and spectral data were used for the isolation and identification of SA. MTT, flow cytometry, in vitro invasion and angiogenesis assays, fluoremetry, ELISA and Real Time qPCR were used to test antimitogenic and chemo-sensitizing activities of SA, cell cycle, apoptosis, proteasome and NFκB-DNA-binding activities, cancer cell invasion and angiogenesis, and expression of cell cycle/apoptosis-related genes. SA showed a time- and dose-dependent (IC₅₀ = 0.95-1.2 mg mL⁻¹) antimitogenic effect against cancer cells with little cytotoxicity on normal fibroblasts (≤20%). SA-altered cell cycle (S/G2-M or G1/G2-M phases) in a time-dependent manner, induced apoptosis, inhibited DNA-binding activity of NFκB (p ≤ 0.0001), chymotrypsin-like/PGPH (peptidyl-glutamyl peptide-hydrolyzing) (p ≤ 0.0001) and the trypsin-like (p ≤ 0.002) activities of 26S proteasome and angiogenesis. SA also differentially sensitized cancer cells to standard chemotherapies with a marked increase in their sensitivity to camptothecin (500-fold), 5FU (20,000-fold), doxorubicin (210-fold), taxol (3134-fold), vinblastine (1000-fold), vincristine (130-fold) and amsacrine (107-fold) compared to standard drugs alone. SA exerted its chemotherapeutic and chemo-sensitizing effects through an array of mechanisms including cell-cycle arrest, apoptosis induction, inhibition of cell proliferation, cell migration, angiogenesis, NFκB DNA-binding and proteasome activities. These results demonstrate the potential of SA as an antimitogenic and chemo-sensitizing agent for human colorectal cancer.

  2. Hsp104 Overexpression Cures Saccharomyces cerevisiae [PSI+] by Causing Dissolution of the Prion Seeds

    Science.gov (United States)

    Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C.

    2014-01-01

    The [PSI+] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI+], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI+]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI+] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI+] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis. PMID:24632242

  3. Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

    Science.gov (United States)

    Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In; Todor, Horia; Ellerbrock, Robyn; Reidy, Michael; Eisenberg, Evan; Masison, Daniel C; Greene, Lois E

    2014-05-01

    The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

  4. Contribution of the protector adsorbed by the active site to the enzyme protection against γ-radiation

    International Nuclear Information System (INIS)

    Kravetskaya, T.P.; Krutyakov, V.M.; Sverdlov, A.G.

    1976-01-01

    It has been shown that 5-methoxytryptamine (mexamine) is a competitive inhibitor of trypsin. This prompted compairing the protection of the enzyme under varying conditions, that is: 90 per cent of the enzyme molecules are in a free state and 90 per cent of the enzyme molecules are bound with the protector molecules. A four-time increase in the protecting effect was observed with the protector bound to the active site of trypsin. A quantitative comparison of the trypsin protection by a tissue extract of a ''cellular'' concentration and by mexamine of a maximum ''organismal'' concentration has shown that radical scavenging is not a principal mechanism responsible for the protector effect in a cell even if the adsorption of the protector on the biopolymers is taken into account

  5. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

    Directory of Open Access Journals (Sweden)

    Scott W. Messenger

    2015-11-01

    Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

  6. The Effect of Gamma radiation, microwave radiation, their interaction and storage on chemical composition, antinutritional factors and the activities of trypsin inhibitor and lipoxygenase of soybean seeds

    Energy Technology Data Exchange (ETDEWEB)

    Abdel-Rahim, E A; Abdel-Fatah, O M [Dept. of Biochem., Faculty of Agric., Cairo University. (Egypt); El-Adawy, M; Badea, M Y [Food Technol. Dept., National Center for Research and Radiation Technol., Atomic Energy Authority (Egypt)

    2000-07-01

    The effect of gamma radiation, microwave radiation, interaction between them; and storage of radiated soybean seeds were investigated to find out the best treatment which had to the maximum reduction of antinutrional factors (Trypsin inhibitor and lipoxygenase activities) without significant effect on the chemical constituents. The gamma rays was used at three doses of 2.5, 5.0 and 8.0 kGy, microwave radiation was at 70 level power for 2 and 4 min; and the storage of seeds was at temperature, R.H. 50-55% for six months. The data revealed that, effects of interaction treatments were more effective than the treatment with microwave or gamma radiation alone.

  7. The Effect of Gamma radiation, microwave radiation, their interaction and storage on chemical composition, antinutritional factors and the activities of trypsin inhibitor and lipoxygenase of soybean seeds

    International Nuclear Information System (INIS)

    Abdel-Rahim, E.A.; Abdel-Fatah, O.M.; El-Adawy, M.; Badea, M.Y.

    2000-01-01

    The effect of gamma radiation, microwave radiation, interaction between them; and storage of radiated soybean seeds were investigated to find out the best treatment which had to the maximum reduction of antinutrional factors (Trypsin inhibitor and lipoxygenase activities) without significant effect on the chemical constituents. The gamma rays was used at three doses of 2.5, 5.0 and 8.0 kGy, microwave radiation was at 70 level power for 2 and 4 min; and the storage of seeds was at temperature, R.H. 50-55% for six months. The data revealed that, effects of interaction treatments were more effective than the treatment with microwave or gamma radiation alone

  8. Ma

    Directory of Open Access Journals (Sweden)

    Ingrid Berthon-Moine

    2012-01-01

    Full Text Available Ma (2009 is a single channel video of a mother and child walking together side by side, holding hands. The title is reminiscent of the affectionate nickname for a mother, 'Ma', but also a concealed way to convey maternal ambivalence. Maternal ambivalence is the result of the tension between the idealisation of motherhood and women’s lived experience of mothering. The maternal struggle finds its source in the difficulty of identifying with the ideological representation of the mother. This image still conveys an idealistic and nostalgic, patriarchal image of maternal love bounded by culture and history. http://podcast.ulcc.ac.uk/accounts/BirkbeckCollege/mamsie/MA.mov

  9. Glycine-rich analogues of Cucurbita maxima trypsin inhibitor (CMTI-III) substituted by valine in position 27 display relatively low antitrypsin activity.

    Science.gov (United States)

    Rózycki, J; Kupryszewski, G; Rolka, K; Ragnarsson, U; Zbytryt, T; Krokoszyńska, I; Otlewski, J

    1993-09-01

    Five new analogues of the trypsin inhibitor CMTI-III were synthesized by the solid-phase method. All analogues containing a valine residue in position 27 and glycine residues in some or all of the positions 9, 11, 14, 17, 19, 29 as well as in two cases a norleucine residue in position 8 displayed association equilibrium constants by 6-7 orders of magnitude lower than the native CMTI-III inhibitor.

  10. Extraction of gelatin from salmon (Salmo salar) fish skin using trypsin-aided process: optimization by Plackett-Burman and response surface methodological approaches.

    Science.gov (United States)

    Fan, HuiYin; Dumont, Marie-Josée; Simpson, Benjamin K

    2017-11-01

    Gelatin from salmon ( Salmo salar ) skin with high molecular weight protein chains ( α -chains) was extracted using trypsin-aided process. Response surface methodology was used to optimise the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gelatin were used as responses in the optimization study. The optimum conditions were determined as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and extraction time at 6 h 16 min. This response surface optimized model was significant and produced an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%). Twofold higher yields of gelatin with high molecular weight protein chains were achieved in the optimized process with trypsin treatment when compared to the process without trypsin.

  11. Effect of condensed tannins in hulls of faba beans (Vivia faba L.) on the activities of trypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1) in digesta collected from the small intestine of pigs

    NARCIS (Netherlands)

    Jansman, A.J.M.; Enting, H.; Verstegen, M.W.A.; Huisman, J.

    1994-01-01

    The effects of condensed tannins in hulls of faba beans (Vicia faba L.) on the activities of trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) in digesta obtained from the small intestine of pigs were studied. Using four castrated male pigs (mean body weight 83 kg) fitted with both a simple

  12. Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

    Directory of Open Access Journals (Sweden)

    Jacky Y Suen

    Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

  13. 18 CFR 385.104 - Rule of construction (Rule 104).

    Science.gov (United States)

    2010-04-01

    ... Definitions § 385.104 Rule of construction (Rule 104). To the extent that the text of a rule is inconsistent with its caption, the text of the rule controls. [Order 376, 49 FR 21705, May 23, 1984] ...

  14. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

    Science.gov (United States)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-05

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Enzyme/Mod. List: E0000000102 [jPOST repository metadata[Archive

    Lifescience Database Archive (English)

    Full Text Available E0000000102 Enzyme1 Trypsin Carbamidomethyl (C) Acetyl (N-term) Oxidation (M) Brucella abortus 104M The Enz...yme specificity was set to trypsin and a maximum of two missed cleavages were allow

  16. Geological constraints on continental arc activity since 720 Ma: implications for the link between long-term climate variability and episodicity of continental arcs

    Science.gov (United States)

    Cao, W.; Lee, C. T.

    2016-12-01

    Continental arc volcanoes have been suggested to release more CO2 than island arc volcanoes due to decarbonation of wallrock carbonates in the continental upper plate through which the magmas traverse (Lee et al., 2013). Continental arcs may thus play an important role in long-term climate. To test this hypothesis, we compiled geological maps to reconstruct the surface distribution of granitoid plutons and the lengths of ancient continental arcs. These results were then compiled into a GIS framework and incorporated into GPlates plate reconstructions. Our results show an episodic nature of global continental arc activity since 720 Ma. The lengths of continental arcs were at minimums during most of the Cryogenian ( 720-670 Ma), the middle Paleozoic ( 460-300 Ma) and the Cenozoic ( 50-0 Ma). Arc lengths were highest during the Ediacaran ( 640-570 Ma), the early Paleozoic ( 550-430 Ma) and the entire Mesozoic with peaks in the Early Triassic ( 250-240 Ma), Late Jurassic-Early Cretaceous ( 160-130 Ma), and Late Cretaceous ( 90-65 Ma). The extensive continental arcs in the Ediacaran and early Paleozoic reflect the Pan-African events and circum-Gondwana subduction during the assembly of the Gondwana supercontinent. The Early Triassic peak is coincident with the final closure of the paleo-Asian oceans and the onset of circum-Pacific subduction associated with the assembly of the Pangea supercontinent. The Jurassic-Cretaceous peaks reflect the extensive continental arcs established in the western Pacific, North and South American Cordillera, coincident with the initial dispersal of the Pangea. Continental arcs are favored during the final assembly and the early-stage dispersal of a supercontinent. Our compilation shows a temporal match between continental arc activity and long-term climate at least since 720 Ma. For example, continental arc activity was reduced during the Cryogenian icehouse event, and enhanced during the Early Paleozoic and Jurassic-Cretaceous greenhouse

  17. Use of the neutron diffraction - H/D exchange technique to determine the conformational dynamics of trypsin

    International Nuclear Information System (INIS)

    Kossiakoff, A.A.

    1982-01-01

    Reported here are studies analyzing the extent and nature of the inherent conformational fluctuations in trypsin by neutron diffraction - hydrogen exchange techniques. The pattern of exchange investigates systematic relationships between exchangeable sites and the structural and chemical properties of the molecule. Our findings that pH 7, 20 0 and 1 year of soaking all sites of trypsin are fully exchanged except those which are especially well protected by the structure. Essentially all the sites in which the peptide hydrogens are bonded directly to water molecules - either in the bulk solvent regions or in interior clusters - are fully exchanged. 41 references, 10 figures

  18. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    International Nuclear Information System (INIS)

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-01-01

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

  19. Digestion of atopic allergens with trypsin α-chymotrypsin and pancreatic kallikrein, and influence of the allergens upon the proteolytic and esterolytic activity of these enzymes

    NARCIS (Netherlands)

    Berrens, L.

    1968-01-01

    The action of bovine trypsin, α-chymotrypsin and pancreatic kallikrein upon a number of atopic allergens has been studied by pH-stat measurements during short-term incubation. Most atopic allergens proved chemically resistant towards these enzymes. Graphs of enzyme susceptibility vs. the ratio of

  20. Changes in antigenicity of porcine serum albumin in gamma-irradiated sausage extract by treatment with pepsin and trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Koth-Bong-Woo-Ri; Song, Eu-Jin [Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 608-737 (Korea, Republic of); Lee, So-Young [Traditional Food Research Group, Korea Food Research Institute, Seongnam 463-746 (Korea, Republic of); Park, Jin-Gyu [Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185 (Korea, Republic of); Lee, Ju-Woon [National Federation of Fisheries Cooperatives, Fisheries Economic Institute, Seoul 138-827 (Korea, Republic of); Byun, Myung-Woo [Department of Culinary Nutrition, Woosong University, Daejon 300-718 (Korea, Republic of); Ahn, Dong-Hyun, E-mail: dhahn@pknu.ac.kr [Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 608-737 (Korea, Republic of)

    2011-11-15

    Pork is known as an allergenic food with porcine serum albumin (PSA, 66 kDa) representing the major allergen. This study was conducted to investigate the change in antigenicity of PSA in gamma-irradiated sausage extract treated with pepsin and trypsin. Sausage products (A and B) were irradiated at 1, 3, 10, and 20 kGy. After irradiation, sausage proteins were extracted and digested with pepsin (1:200, 30 min) and trypsin (1:300, 5, 30, 60, 90, and 120 min). The binding ability of PSA in extracts of the irradiated sausages (A and B) decreased by over 3 kGy relative to the binding ability of PSA in extracts of intact sausages and showed no notable differences when the dose of radiation ranged from 3 to 20 kGy. After treatment with pepsin and trypsin, the binding ability of PSA in extracts of the irradiated sausages was decreased more relative to that of intact sausages and showed no significant differences when the period of trypsin treatment is increased or when the dose of irradiation is increased. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that there was no visible change in the intensity of the PSA band in extracts of the irradiated sausages. After pepsin and trypsin treatment, the intensity of PSA band faded with increasing doses of irradiation. In conclusion, antigenicity of PSA in pork sausages could be reduced by gamma irradiation. - Highlights: > Change in antigenicity of PSA in irradiated sausage extract (ISE) was examined. > Binding ability of PSA in ISE was decreased compared to intact extract. > Binding ability of PSA in ISE after enzyme treatments was also further decreased. > Intensity of PSA band in ISE after enzyme treatments became weak.

  1. Changes in antigenicity of porcine serum albumin in gamma-irradiated sausage extract by treatment with pepsin and trypsin

    International Nuclear Information System (INIS)

    Kim, Koth-Bong-Woo-Ri; Song, Eu-Jin; Lee, So-Young; Park, Jin-Gyu; Lee, Ju-Woon; Byun, Myung-Woo; Ahn, Dong-Hyun

    2011-01-01

    Pork is known as an allergenic food with porcine serum albumin (PSA, 66 kDa) representing the major allergen. This study was conducted to investigate the change in antigenicity of PSA in gamma-irradiated sausage extract treated with pepsin and trypsin. Sausage products (A and B) were irradiated at 1, 3, 10, and 20 kGy. After irradiation, sausage proteins were extracted and digested with pepsin (1:200, 30 min) and trypsin (1:300, 5, 30, 60, 90, and 120 min). The binding ability of PSA in extracts of the irradiated sausages (A and B) decreased by over 3 kGy relative to the binding ability of PSA in extracts of intact sausages and showed no notable differences when the dose of radiation ranged from 3 to 20 kGy. After treatment with pepsin and trypsin, the binding ability of PSA in extracts of the irradiated sausages was decreased more relative to that of intact sausages and showed no significant differences when the period of trypsin treatment is increased or when the dose of irradiation is increased. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that there was no visible change in the intensity of the PSA band in extracts of the irradiated sausages. After pepsin and trypsin treatment, the intensity of PSA band faded with increasing doses of irradiation. In conclusion, antigenicity of PSA in pork sausages could be reduced by gamma irradiation. - Highlights: → Change in antigenicity of PSA in irradiated sausage extract (ISE) was examined. → Binding ability of PSA in ISE was decreased compared to intact extract. → Binding ability of PSA in ISE after enzyme treatments was also further decreased. → Intensity of PSA band in ISE after enzyme treatments became weak.

  2. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Highsmith, R.F.; Gallaher, M.J.

    1986-03-05

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

  3. Frequency of inhibitors of daphnid trypsin in the widely distributed cyanobacterial genus Planktothrix

    DEFF Research Database (Denmark)

    Rohrlack, T.; Christoffersen, K.; Friberg-Jensen, U.

    2005-01-01

    on the frequency of such compounds in the widely distributed cyanobacterial genus Planktothrix. Of the 89 Planktothrix strains analysed, about 70% produced inhibitors of daphnid trypsin. The strains tested positive represented three common Planktothrix species and were isolated from diverse localities...

  4. BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells

    Science.gov (United States)

    Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity ...

  5. Rapid tryptic mapping using enzymatically active mass spectrometer probe tips

    Energy Technology Data Exchange (ETDEWEB)

    Dogruel, D.; Williams, P.; Nelson, R.W. [Arizona State Univ., Tempe, AZ (United States)

    1995-12-01

    A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. 19 refs., 6 figs., 2 tabs.

  6. Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability.

    Science.gov (United States)

    Ketnawa, Sunantha; Benjakul, Soottawat; Martínez-Alvarez, Oscar; Rawdkuen, Saroat

    2017-01-15

    The peptidase from the viscera of farmed giant catfish was used for producing gelatin hydrolysates (HG) and compared with those produced from commercial bovine trypsin (HB). The degree of hydrolysis (DH) observed suggests that proteolytic cleavage rapidly occurred within the first 120min of incubation, and there was higher DH in HG than in HB. HG demonstrated the highest ACE-inhibitory activity, DPPH, ABTS radical scavenging activity, and FRAP. HB showed the highest FRAP activity. The DPPH radical scavenging activity of HG was quite stable over the pH range of 1-11, but it increased slightly when the heating duration time reached 240min at 100°C. The ACE-inhibitory activity of HG showed the highest stability at a pH of 7, and it remained very stable at 100°C for over 15-240min. The visceral peptidase from farmed giant catfish could be an alternative protease for generating protein hydrolysates with desirable bioactivities. The resulting hydrolysates showed good stability, making them potential functional ingredients for food formulations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. The Prevalence of Ionized Gas Outflow Signatures in SDSS-IV MaNGA Active Galactic Nuclei

    Science.gov (United States)

    Flores, Anthony M.; Wylezalek, Dominika; Zakamska, Nadia

    2018-01-01

    Actively accreting supermassive black holes (AGN) can have a variety of effects on their host galaxies, from generating large regions of hot, photoionized gas, to driving AGN feedback in the form of galaxy wide outflows that may affect the evolution of the galaxy over time by quenching their star formation and by thus setting limits to the total mass of their host galaxy. The focus of this work is to assess the prevalence of AGN-driven outflows in low-redshift AGN of moderate power using IFU observations of 2778 galaxies available through SDSS-IV MaNGA.SDSS-IV MaNGA is an optical spectroscopic IFU survey which will have obtained spatially resolved spectroscopic observations of ~10,000 galaxies at z ≤ 0.1 and with stellar masses >10^9 solar masses over the next three years, allowing us to describe the kinematic properties of a large galaxy sample across different spatial regions.We have re-mapped the kinematics of the [O III] emission line to account for asymmetries and secondary kinematic components in the emission line brought on by potential AGN-driven outflows. Using all galaxies currently in the MaNGA survey, we implement a new fitting procedure to help determine the prevalence of these secondary components. Specifically, we use the non-parametric W80 value as a proxy for velocity dispersion, which we expect to be affected especially in the case of asymmetries and broadening of the emission lines. Separating these galaxies into two samples of independently identified AGN candidates and non-AGN, I will show that broad secondary components are twice as common in MaNGA-selected AGN compared to galaxies in MaNGA not classified as AGN. Moreover, when the underlying distribution of W80 values are compared between samples, I will show that the differences in these distributions are statistically significant. This demonstrates that large IFU survey like SDSS-IV MaNGA will uncover many previously unknown AGN and AGN feedback signatures. Outflows and feedback from low

  8. Comparison of [18F]FLT and [18F]FDG in in vitro cancer cell uptake and glucose effect

    International Nuclear Information System (INIS)

    Soo Jung Lim; Jin-Sook Ryu; Heuiran Lee; Seok Young Kim; Seung Jun Oh; Dae Hyuk Moon

    2004-01-01

    [18F]FLT is a new radiopharmaceutical for cell proliferation. We compared [18F]FLT and [18F]FDG in in vitro cancer cell uptake and glucose effect. Method: In vitro cancer cell uptake of [18F]FLT was evaluated using SCC7(mouse squamous cell carcinoma). At 24 hours after seeding 1 x 106 cells/well in 6 well plates with RPMI 1640 medium, culture media were changed to medium with glucose free or glucose concentration of 100 mg/dl. Then, [18F]FLT 5 μCi/50 ml was added to each well. After incubation for 30, 60, 90, 120 minutes, cells were washed twice by PBS, and harvested using 0.25% trypsin-EDTA. After centrifugation and counting at gamma counter, cell uptake was calculated by % activity of cellular uptake to total activity of cell and supernatant. For comparison, same tumor cell uptake experiment was performed with [18F]FDG. Results: After incubation with SCC7 cell line for 30, 60, 90, 120 minutes, [18F]FLT showed 1.95%, 2.17%, 2.10% and 2.80% of cell uptake in glucose free media, respectively. The results [18F]FLT uptake in glucose 100 mg/dl media were 1.82%, 1.87%, 1.97%, and 2.94%, respectively. The results of [18F]FDG in glucose free media were 2.50%, 3.47%, 5.04%, and 10.4%, whereas those in glucose 100 mg/dl media were 1.60%, 1.79%, 1.53%, and 1.82%, respectively. Conclusion: In contrast to [18F]FDG, [18F]FLT uptake in cancer cell was not affected by glucose concentration. In physiologic glucose concentration, [18F]FLT uptake in SCC7 cell line was significantly higher than [18F]FDG uptake after 120 minutes incubation. In [18F]FLT PET imaging may not need fasting for preparation before imaging study. (authors)

  9. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    Science.gov (United States)

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  10. Solid KHT tumor dispersal for flow cytometric cell kinetic analysis

    International Nuclear Information System (INIS)

    Pallavicini, M.G.; Folstad, L.J.; Dunbar, C.

    1981-01-01

    A bacterial neutral protease was used to disperse KHT solid tumors into single cell suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral protease disaggregation under conditions which would be suitable for routine tumor dispersal was compared with a trypsin/DNase procedure. Cell yield, clonogenic cell survival, DNA distributions of untreated and drug-perturbed tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated. Tumors dispersed with neutral protease yielded approximately four times more cells than those dispersed with trypsin/DNase and approximately a 1.5-fold higher plating efficiency in a semisolid agar system. Quantitative analysis of DNA distributions obtained from untreated and cytosine-arabinoside-perturbed tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated thymidine during the cell cycle were also similar with neutral protease and trypsin/DNase dispersal. Preferential phase-specific cell loss was not obseved with either technique. We find that neutral protease provides good single cell suspensions of the KHT tumor for cell survival measurements and for cell kinetic analysis of drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required

  11. 50 CFR 17.104 - Prohibitions.

    Science.gov (United States)

    2010-10-01

    ... PLANTS (CONTINUED) ENDANGERED AND THREATENED WILDLIFE AND PLANTS (CONTINUED) Manatee Protection Areas § 17.104 Prohibitions. Except as provided in § 17.105, (a) Manatee sanctuary. It is unlawful for any person to engage in any waterborne activity within a manatee sanctuary. (b) Manatee refuge. It is...

  12. Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents.

    Science.gov (United States)

    Pazhang, Mohammad; Mehrnejad, Faramarz; Pazhang, Yaghub; Falahati, Hanieh; Chaparzadeh, Nader

    2016-01-01

    The effect of glycerol and sorbitol on the stability of porcine pancreas trypsin was investigated in this work. Molecular dynamics simulation and thermostability results showed that trypsin has two flexible regions, and polyols (sorbitol and glycerol) stabilize the enzyme by decreasing the flexibility of these regions. Radial distribution function results exhibited that sorbitol and glycerol were excluded from the first water layer of the enzyme, therefore decrease the flexibility of the regions by preferential exclusion. Also, results showed that the stabilization effect of sorbitol is more than glycerol. This observation could be because of the larger decrease in the fluctuations of trypsin in the presence of sorbitol. We also examined the role of solvent's hydrophobicity in enzyme stabilization by sorbitol and glycerol. To do so, the thermostability of trypsin was evaluated in the presence of solvents with different hydrophobicity (methanol, ethanol, isopropanol and n-propanol) in addition to the polyols. Our results depicted that glycerol is a better stabilizer than sorbitol in the presence of hydrophobic solvents (n-propanol), whereas sorbitol is a better stabilizer than glycerol in the presence of hydrophilic solvents (methanol). © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  13. Conformational Network and Residence Time Estimation of Trypsin-Benzamidine Unbinding Pathways

    OpenAIRE

    Dickson, Alex; Lotz, Samuel D.

    2016-01-01

    In this poster we present results from molecular dynamics sampling of benzamidine unbinding from trypsin. We give background on the weighted ensemble technique used (WExplore) and the Markovian state model construction. Our network shows three unique unbinding pathways including a never before observed unbinding pathway. We also estimate residence time to within one order of magnitude to the experimental value.

  14. Radioimmunoassay of trypsin in dried blood importance for the neonatal detection of cystic fibrosis

    International Nuclear Information System (INIS)

    Travert, G.; Mustin, C.; Fernandez, Y.

    1981-01-01

    The demonstration of very high levels of immunoreactive trypsin in the blood of newborn infants with cystic fibrosis has provided a new way of detecting the disease soon after birth. A radioimmunoassay of trypsin in the eluate of blood dried on filter paper has now been developed. The sensitivity and accuracy of the method, as well as the good correlation observed between the values obtained and those of the conventional plasma assay, indicate that it is reliable and well adapted to the newborn. The new assay can easily be inserted into the present system of neonatal disease detection. A preliminary assessment of more than 5000 tests enables the authors to report an early diagnosis of proven cystic fibrosis and to discuss an essential aspect of mass-detection methods: the indicence of false-positive results [fr

  15. Potent Anti-Inflammatory Activity of Pyrenocine A Isolated from the Marine-Derived Fungus Penicillium paxilli Ma(G)K

    Science.gov (United States)

    Toledo, Thaís Regina; Dejani, Naiara N.; Monnazzi, Luis Gustavo Silva; Kossuga, Miriam H.; Berlinck, Roberto G. S.; Sette, Lara D.; Medeiros, Alexandra I.

    2014-01-01

    Very little is known about the immunomodulatory potential of secondary metabolites isolated from marine microorganisms. In the present study, we characterized pyrenocine A, which is produced by the marine-derived fungus Penicillium paxilli Ma(G)K and possesses anti-inflammatory activity. Pyrenocine A was able to suppress, both pretreatment and posttreatment, the LPS-induced activation of macrophages via the inhibition of nitrite production and the synthesis of inflammatory cytokines and PGE2. Pyrenocine A also exhibited anti-inflammatory effects on the expression of receptors directly related to cell migration (Mac-1) as well as costimulatory molecules involved in lymphocyte activation (B7.1). Nitrite production was inhibited by pyrenocine A in macrophages stimulated with CpG but not Poly I:C, suggesting that pyrenocine A acts through the MyD88-dependent intracellular signaling pathway. Moreover, pyrenocine A is also able to inhibit the expression of genes related to NFκB-mediated signal transduction on macrophages stimulated by LPS. Our results indicate that pyrenocine A has promissory anti-inflammatory properties and additional experiments are necessary to confirm this finding in vivo model. PMID:24574582

  16. Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion.

    Science.gov (United States)

    Guo, Cong; Zhao, Xinyuan; Zhang, Wanjun; Bai, Haihong; Qin, Weijie; Song, Haifeng; Qian, Xiaohong

    2017-08-01

    Highly efficient protein digestion is one of the key issues in the "bottom-up" strategy-based proteomic studies. Compared with the time-consuming solution-based free protease digestion, immobilized protease digestion offers a promising alternative with obviously improved sample processing throughput. In this study, we proposed a new immobilized protease digestion strategy using two kinds of polymer-grafted graphene oxide (GO) conjugated trypsin. The polymer brush grafted GO was prepared using in situ polymer growth on initiator-functionalized GO using surface-initiated atom transfer radical polymerization (SI-ATRP) and characterized by AFM, TEM, TGA, and XPS. The polymer brush grafted GO supports three-dimensional trypsin immobilization, which not only increases the loading amount but also improves accessibility towards protein substrates. Both of the two types of immobilized trypsin provide 700 times shorter digestion time, while maintaining comparable protein/peptide identification scale compared with that of free trypsin digestion. More interestingly, combined application of the two types of immobilized trypsin with different surface-grafted polymers leads to at least 18.3/31.3% enhancement in protein/peptide identification compared with that obtained by digestion using a single type, indicating the potential of this digestion strategy for deeper proteome coverage using limited mass spectrometer machine hour. We expect these advantages may find valuable application in high throughput clinical proteomic studies, which often involve processing of a large number of samples. Graphical abstract Preparation of polymer brushes grafted and trypsin immobilized graphene oxide and its application in proteome digestion and mass spectrometry identification.

  17. DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

    Science.gov (United States)

    Frouco, Gonçalo; Freitas, Ferdinando B; Coelho, João; Leitão, Alexandre; Martins, Carlos; Ferreira, Fernando

    2017-06-15

    African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently

  18. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

    Science.gov (United States)

    Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata

    NARCIS (Netherlands)

    Glawe, G.A.; Zavala, J.A.; Kessler, A.; Van Dam, N.M.; Baldwin, I.T.

    2003-01-01

    Genotypes of the wild tobacco Nicotiana attenuata from different geographic regions in North America vary considerably in the level of constitutive and inducible trypsin protease inhibitors (TrypPIs), a potent direct defense, as well as in the production of herbivore-induced volatiles that function

  20. Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

    Directory of Open Access Journals (Sweden)

    M.T. Pereira

    2005-11-01

    Full Text Available Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05. Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp, a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively. The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

  1. Evaluation of two different protocols for peripheral blood stem cell collection with the Fresenius AS 104 blood cell separator.

    Science.gov (United States)

    Menichella, G; Lai, M; Pierelli, L; Vittori, M; Serafini, R; Ciarli, M; Foddai, M L; Salerno, G; Sica, S; Scambia, G; Leone, G; Bizzi, B

    1997-01-01

    Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant. We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program. Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures. High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.

  2. The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes.

    Science.gov (United States)

    Bode, W; Greyling, H J; Huber, R; Otlewski, J; Wilusz, T

    1989-01-02

    The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.

  3. Substrate Discrimination by ClpB and Hsp104

    Directory of Open Access Journals (Sweden)

    Danielle M. Johnston

    2017-05-01

    Full Text Available ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

  4. Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

    Directory of Open Access Journals (Sweden)

    Rebrikov Denis V

    2004-01-01

    Full Text Available Abstract Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa and 226 amino acid residues (Mcalc 23.5 kDa, respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70% and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%. The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases.

  5. Phenolic compounds from Viscum album tinctures enhanced antitumor activity in melanoma murine cancer cells.

    Science.gov (United States)

    Melo, Michelle Nonato de Oliveira; Oliveira, Adriana Passos; Wiecikowski, Adalgisa Felippe; Carvalho, Renato Sampaio; Castro, Juliana de Lima; de Oliveira, Felipe Alves Gomes; Pereira, Henrique Marcelo Gualberto; da Veiga, Venicio Feo; Capella, Marcia Marques Alves; Rocha, Leandro; Holandino, Carla

    2018-03-01

    Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe ( Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

  6. SDSS-IV MaNGA: Galaxy Pair Fraction and Correlated Active Galactic Nuclei

    Science.gov (United States)

    Fu, Hai; Steffen, Joshua L.; Gross, Arran C.; Dai, Y. Sophia; Isbell, Jacob W.; Lin, Lihwai; Wake, David; Xue, Rui; Bizyaev, Dmitry; Pan, Kaike

    2018-04-01

    We have identified 105 galaxy pairs at z ∼ 0.04 with the MaNGA integral-field spectroscopic data. The pairs have projected separations between 1 and 30 kpc, and are selected to have radial velocity offsets less than 600 km s‑1 and stellar mass ratio between 0.1 and 1. The pair fraction increases with both the physical size of the integral-field unit and the stellar mass, consistent with theoretical expectations. We provide the best-fit analytical function of the pair fraction and find that ∼3% of M* galaxies are in close pairs. For both isolated galaxies and paired galaxies, active galactic nuclei (AGNs) are selected using emission-line ratios and Hα equivalent widths measured inside apertures at a fixed physical size. We find AGNs in ∼24% of the paired galaxies and binary AGNs in ∼13% of the pairs. To account for the selection biases in both the pair sample and the MaNGA sample, we compare the AGN comoving volume densities with those expected from the mass- and redshift-dependent AGN fractions. We find a strong (∼5×) excess of binary AGNs over random pairing and a mild (∼20%) deficit of single AGNs. The binary AGN excess increases from ∼2× to ∼6× as the projected separation decreases from 10–30 to 1–10 kpc. Our results indicate that the pairing of galaxies preserves the AGN duty cycle in individual galaxies but increases the population of binary AGNs through correlated activities. We suggest tidally induced galactic-scale shocks and AGN cross-ionization as two plausible channels to produce low-luminosity narrow-line-selected binary AGNs.

  7. Proton NMR studies of Cucurbita maxima trypsin inhibitors: Evidence for pH-dependent conformational change and his25 - try27 interaction

    Energy Technology Data Exchange (ETDEWEB)

    Krishnamoorthi, R.; Chanlan Sun Lin; Yuxi Gong (Kansas State Univ., Manhattan (United States)); VanderVelde, D. (Univ. of Kansas, Lawrence (United States)); Hahn, K. (Univ. of Colorado, Denver (United States))

    1992-01-28

    A pH-dependent His25-Tyr27 interaction was demonstrated in the case of Cucurbita maxima trypsin inhibitors (CMTI-I and CMTI-III) by means of nuclear magnetic resonance (NMR) spectroscopy. pH titration, line widths, peak shapes, deuterium exchange kinetics, and two-dimensional nuclear Overhauser effect spectroscopy (NOESY) were employed to characterize a conformational change involving Tyr27, which was shown to be triggered by deprotonation of His25 around pH 6. A hydrogen bond is proposed to be formed between N{sub {epsilon}} of His25 and OH of Tyr27, as a distance between the atoms, His25 N{epsilon} and Tyr25 OH, of 3.02 {angstrom} is consistent with a model built with NOE-derived distance constraints. The presently characterized relative orientations of His25 and Tyr27 are of functional significance, as these residues make contact with the enzyme in the enzyme-inhibitor complex. Furthermore, trypsin assay and inhibitor-binding studies showed that conformations of trypsin and the squash inhibitor complex. Furthermore, trypsin assay and inhibitor-binding studies showed that conformations of trypsin and the squash inhibitor were functionally relevant only in the pH range 6-8. The pK{sub a} of His25 was determined and found to be influenced by Glu9/Lys substitution and by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6. As these sites are located far (>10 {angstrom}) from His25, the results point out conformational changes that are propagated to a distant site in the protein molecule.

  8. Alumni careers : MA Comparative European Social Studies Zuyd University

    NARCIS (Netherlands)

    Albert Riga

    2004-01-01

    In the preparation of the celebration of the 10th anniversary of the MA CESS all alumni were invited to participate in an alumni survey. The purpose of the survey was to thoroughly investigate the professional activities of MA CESS alumni and the way they build an alumni community. Another reason

  9. Methamphetamine-induced neurotoxicity linked to UPS dysfunction and autophagy related changes that can be modulated by PKCδ in dopaminergic neuronal cells

    Science.gov (United States)

    Lin, Mengshien; Shivalingappa, Prashanth Chandramani; Jin, Huajun; Ghosh, Anamitra; Anantharam, Vellareddy; Ali, Syed; Kanthasamy, Anumantha G.; Kanthasamy, Arthi

    2012-01-01

    A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and UPS dysfunction are not well understood. The present study investigates the contributions of PKC delta (PKCδ) mediated signaling events in MA-induced autophagy, UPS dysfunction and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased of PKCδ activation, caspase-3 activation, accumulation of ubiquitin positive aggregates and microtubule associated light chain-3 (LC3-II) levels. Interestingly, siRNA mediated knockdown of PKCδ or overexpression of cleavage resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3 mediated apoptotic cell death in the nigrostriatal dopaminergic

  10. Alumni careers : MA Comparative European Social Studies Zuyd University

    OpenAIRE

    Riga, Albert

    2004-01-01

    In the preparation of the celebration of the 10th anniversary of the MA CESS all alumni were invited to participate in an alumni survey. The purpose of the survey was to thoroughly investigate the professional activities of MA CESS alumni and the way they build an alumni community. Another reason for starting the project is that it may play a part in maintaining and enhancing a mutually beneficial relationship between MA CESS and its alumni. All over Europe there are graduates working in the ...

  11. Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

    Directory of Open Access Journals (Sweden)

    Maarke J.E. Roelofs

    2014-01-01

    Full Text Available Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO, fluconazole (FLUC, flusilazole (FLUS, hexaconazole (HEXA, myconazole (MYC, penconazole (PEN, prochloraz (PRO, tebuconazole (TEBU, triadimefon (TRIA, and triticonazole (TRIT were examined using murine Leydig (MA-10 cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM or TEBU (IC50 = 2.4 μM in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM and effect potencies (REPs were calculated relative to the known AR antagonist flutamide (FLUT. FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61 and MYC the least potent (REP = 0.03 AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human risk assessment of this class of compounds.

  12. Industrial chains and innovation activity in Małopolska region in 2008-2010

    Directory of Open Access Journals (Sweden)

    Świadek Arkadiusz

    2014-05-01

    Full Text Available Znaczenie powiązań przemysłowych dla aktywności innowacyjnej przedsiębiorstw w regionie małopolskim w latach 2008-2010 Głównym celem prowadzonych badań była próba poszukiwania zmiennych warunków wpływu łańcuchów dostaw na działalność innowacyjną przedsiębiorstw w obrębie wybranego regionalnego systemu przemysłowego, a tym samym określenie warunków brzegowych dla modelu regionalnej sieci innowacji, biorąc pod uwagę specyfikę województwa małopolskiego. Badanie zostało oparte na kwestionariuszu ankietowym i wykonane na grupie 500 firm z Małopolski. W badaniu wykorzystano modelowanie probitowe. Metoda ta jest skutecznym narzędziem analizy dużych, ale statycznych prób, w których zmienna zależna ma charakter jakościowy.

  13. Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

    DEFF Research Database (Denmark)

    Klukkert, Marten; Van De Weert, Marco; Fanø, Mathias

    2015-01-01

    was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially...... reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied...... compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs....

  14. Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization

    Directory of Open Access Journals (Sweden)

    Tsuey-Ming Chen

    2016-03-01

    Full Text Available The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG, stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

  15. Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation

    Directory of Open Access Journals (Sweden)

    Paula A. Baldión

    2018-01-01

    Full Text Available Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1, and OLC expanded after trypsinization (EXP-21 were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.

  16. Zhigang Ma

    Indian Academy of Sciences (India)

    Home; Journals; Bulletin of Materials Science. Zhigang Ma. Articles written in Bulletin of Materials Science. Volume 35 Issue 4 August 2012 pp 575-578. Degradation and miscibility of poly(DL-lactic acid)/poly(glycolic acid) composite films: Effect of poly(DL-lactic-co-glycolic acid) · Zhigang Ma Na Zhao Chengdong Xiong.

  17. APC senses cell-cell contacts and moves to the nucleus upon their disruption.

    Science.gov (United States)

    Brocardo, M G; Bianchini, M; Radrizzani, M; Reyes, G B; Dugour, A V; Taminelli, G L; Gonzalez Solveyra, C; Santa-Coloma, T A

    2001-06-22

    The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored. Copyright 2001 Academic Press.

  18. Synthesis and Crystal Structure of an Unprecedented Supramolecular Complex[Co(μ2-ClO4)2(H2O)2]·2MA

    Institute of Scientific and Technical Information of China (English)

    XU,Jing; BAI,Zhengshuai; SUN,Weiyin

    2009-01-01

    A new supramolecular framework[Co(μ2-C104)2(H2O)2]·2MA(1)[MA=melamine(C3H6N6)]has been syn-thesized by a hydrothermal method.Interestingly,there ale inorganic and organic building blocks with two different supramolecular synthons:(a)2D(4,4)network constructed by infinite inorganic 1D chains through interchain hy-drogen bonding interactions;(b)1D zigzag organic chains formed by hydrogen bonds, which further stack up through,ππ-interactions between the two adjacent MA molecules.The entire structure of 1 is a 3D supramolecular framework resulting from the presence of abundant hydrogen bonds between infinite[CO(μ2-C1O4)2(H2O)2]n chains and zigzag MA chains in different sheets.1 gives a nice example of supramolecular framework based on non-covalent interactions including hydrogen bonding and π-π interactions.

  19. Human and bovine spinal disc mechanics subsequent to trypsin injection

    Directory of Open Access Journals (Sweden)

    Jeremy Alsup

    2017-10-01

    The Translational Potential of this Article: Preclinical testing of novel spinal devices is essential to the design validation and regulatory processes, but current testing techniques rely on cadaveric testing of primarily older spines with essentially random amounts of disc degeneration. The present work investigates the viability of using trypsin injections to create a more uniform preclinical model of disc degeneration from a mechanics perspective, for the purpose of testing spinal devices. Such a model would facilitate translation of new spinal technologies to clinical practice.

  20. Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

    Science.gov (United States)

    Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

    2016-12-01

    Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be

  1. Ma ma (2015. A reflection of the Spanish woman with breast cáncer

    Directory of Open Access Journals (Sweden)

    Clara PERALTA NUENO

    2017-12-01

    Full Text Available Ma ma (2015 is a Spanish drama film directed by Julio Medem and produced by Morena Films. This film tells the story of Magda’s life, whose character is interpreted by Penélope Cruz. After being diagnosed with a breast cancer, Magda, an unemployed teacher, will not know how to act nor how this disease will change her life. But little by little, she will bring into the surface all her vital energy. Breast cancer represents the most frequent type of cancer among women. Although the majority of the identified risk factors can be modified, the health systems focus on activities that let strengthen the primary prevention and activities of secondary and tertiary prevention. The early detection and the right treatment are essential to fight against this disease. The emotional process caused by the presence of cancer involves a mechanism of psychic reconstitution that allows the patient develop an answer to the organic, cognitive, social, and emotional challenges they have to deal with. This process is easier when they have a solid socio-family support network among other factors.

  2. Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure.

    Science.gov (United States)

    Cai, M; Gong, Y; Prakash, O; Krishnamoorthi, R

    1995-09-26

    Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI-V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence-specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI-V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Clinical analysis of anti-Ma2-associated encephalitis.

    Science.gov (United States)

    Dalmau, Josep; Graus, Francesc; Villarejo, Alberto; Posner, Jerome B; Blumenthal, Deborah; Thiessen, Brian; Saiz, Albert; Meneses, Patricio; Rosenfeld, Myrna R

    2004-08-01

    Increasing experience indicates that anti-Ma2-associated encephalitis differs from classical paraneoplastic limbic or brainstem encephalitis, and therefore may be unrecognized. To facilitate its diagnosis we report a comprehensive clinical analysis of 38 patients with anti-Ma2 encephalitis. Thirty-four (89%) patients presented with isolated or combined limbic, diencephalic or brainstem dysfunction, and four with other syndromes. Considering the clinical and MRI follow-up, 95% of the patients developed limbic, diencephalic or brainstem encephalopathy. Only 26% had classical limbic encephalitis. Excessive daytime sleepiness affected 32% of the patients, sometimes with narcolepsy-cataplexy and low CSF hypocretin. Additional hormonal or MRI abnormalities indicated diencephalic-hypothalamic involvement in 34% of the patients. Eye movement abnormalities were prominent in 92% of the patients with brainstem dysfunction, but those with additional limbic or diencephalic deficits were most affected; 60% of these patients had vertical gaze paresis that sometimes evolved to total external ophthalmoplegia. Three patients developed atypical parkinsonism, and two a severe hypokinetic syndrome with a tendency to eye closure and dramatic reduction of verbal output. Neurological symptoms preceded the tumour diagnosis in 62% of the patients. Brain MRI abnormalities were present in 74% of all patients and 89% of those with limbic or diencephalic dysfunction. Among the 34 patients with cancer, 53% had testicular germ-cell tumours. Two patients without evidence of cancer had testicular microcalcification and one cryptorchidism, risk factors for testicular germ-cell tumours. After neurological syndrome development, 17 of 33 patients received oncological treatment (nine also immunotherapy), 10 immunotherapy alone, and six no treatment. Overall, 33% of the patients had neurological improvement, three with complete recovery; 21% had long-term stabilization, and 46% deteriorated. Features

  4. Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

    Science.gov (United States)

    Zhang, Ming; Sun, Bo; Zhang, Qi; Gao, Rong; Liu, Qiao; Dong, Fangting; Fang, Haiqin; Peng, Shuangqing; Li, Famei; Yan, Xianzhong

    2017-01-15

    A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating

    International Nuclear Information System (INIS)

    Haustermans, K.; Hofland, I.; Ramaekers, M.; Ivanyi, D.; Balm, A.J.M.; Geboes, K.; Lerut, T.; Schueren, E. van der; Begg, A.C.

    1996-01-01

    Purpose: To determine the feasibility of using cytokeratin antibodies to distinguish normal and malignant cells in human tumors using flow cytometry. The goal was ultimately to increase the accuracy of cell kinetic measurements on human tumor biopsies. Material and methods: A panel of four antibodies was screened on a series of 48 tumors from two centres; 22 head and neck tumors (Amsterdam) and 26 esophagus carcinomas (Leuven). First, screening was carried out by immunohistochemistry on frozen sections to test intensity of staining and the fraction of cytokeratin-positive tumor cells. The antibody showing the most positive staining was then used for flow cytometry on the same tumor. Results: The two broadest spectrum antibodies (AE1/AE3, E3/C4) showed overall the best results with immunohistochemical staining, being positive in over 95% of tumors. Good cell suspensions for DNA flow cytometry could be made from frozen material by a mechanical method, whereas enzymatic methods with trypsin or collagenase were judged failures in almost all cases. >From fresh material, both collagenase and trypsin produced good suspensions for flow cytometry, although the fraction of tumor cells, judged by proportion aneuploid cells, was markedly higher for trypsin. Using the best cytokeratin antibody for each tumor, two parameter flow cytometry was done (cytokeratin versus DNA content). Enrichment of tumor cells was then tested by measuring the fraction of aneuploid cells (the presumed malignant population) of cytokeratin-positive cells versus all cells. An enrichment factor ranging between 0 (no enrichment) and 1 (perfect enrichment, tumor cells only) was then calculated. The average enrichment was 0.60 for head and neck tumors and 0.59 for esophagus tumors. Conclusions: We conclude that this method can substantially enrich the proportion of tumor cells in biopsies from carcinomas. Application of this method could significantly enhance accuracy of tumor cell kinetic measurements

  6. Effect of protracted whole-body gamma irradiation with 6.7 Gy and 4.8 Gy (700 and 500 R) on trypsin inhibition activity of blood, cervical mucus and on morphological structure of cervix in ewes

    International Nuclear Information System (INIS)

    Molnarova, M.; Arendarcik, J.; Molnar, P.

    1984-01-01

    The pattern of changes in the trypsin inhibition activities (TIA) of blood plasma, cervical mucus and the morphological structure of the cervix was studied in ewes exposed to 60 Co radiation for seven and five days, the radiation doses being 6.7 Gy and 4.8 Gy, respectively. During exposure, the group of ewes irradaited with 4.8 Gy was given the Roboran vitamin addition and following irradiation ampicillin (5250 mg). TIA was determined from retardation of the hydrolysis of the synthetic substrate N-alpha-tosyl-p-nitroanilide by bovine trypsin; the TIA was expressed as the percentage of inhibited trypsin. Almost all the studied TIA values of blood plasma and cervical mucus were increased in the irradiated animals, the range being from 103.1 to 155.0% of the levels for non-irradiated ewes. A reduction was recorded only in the total TIA of blood plasma in the group irradiated with a dose of 6.7 Gy (83.1% of the values for non-irradiated animals). In the group of animals irradiated with 4.8 Gy and non Roboran administered, the TIA of cervical mucus was observed to decrease to 92.4%. It was found during the study of changes in the proportion of glands in the stroma and changes in epithelium thickness in the mucous membrane of the cervix uteri that the irradiated ewes had the epithelium thickness reduced to 95.3% to 65.5% and that their stromal gland number decreased to 75.4% to 79.7% of that recorded in non-irradiated animals. It was only in the group given the Roboran supplement that an increase to 123.7% of the gland number for untreated ewes was recorded on the tenth day after termination of the irradiation

  7. AVCRI104P3, a novel multitarget compound with cognition-enhancing and anxiolytic activities: studies in cognitively poor middle-aged mice.

    Science.gov (United States)

    Giménez-Llort, L; Ratia, M; Pérez, B; Camps, P; Muñoz-Torrero, D; Badia, A; Clos, M V

    2015-06-01

    The present work describes, for the first time, the in vivo effects of the multitarget compound AVCRI104P3, a new anticholinesterasic drug with potent inhibitory effects on human AChE, human BuChE and BACE-1 activities as well as on the AChE-induced and self-induced Aβ aggregation. We characterized the behavioral effects of chronic treatment with AVCRI104P3 (0.6 μmol kg(-1), i.p., 21 days) in a sample of middle aged (12-month-old) male 129/Sv×C57BL/6 mice with poor cognitive performance, as shown by the slow acquisition curves of saline-treated animals. Besides, a comparative assessment of cognitive and non-cognitive actions was done using its in vitro equipotent doses of huprine X (0.12 μmol kg(-1)), a huperzine A-tacrine hybrid. The screening assessed locomotor activity, anxiety-like behaviors, cognitive function and side effects. The results on the 'acquisition' of spatial learning and memory show that AVCRI104P3 exerted pro-cognitive effects improving both short- and long-term processes, resulting in a fast and efficient acquisition of the place task in the Morris water maze. On the other hand, a removal test and a perceptual visual learning task indicated that both AChEIs improved short-term 'memory' as compared to saline treated mice. Both drugs elicited the same response in the corner test, but only AVCRI104P3 exhibited anxiolytic-like actions in the dark/light box test. These cognitive-enhancement and anxiolytic-like effects demostrated herein using a sample of middle-aged animals and the lack of adverse effects, strongly encourage further studies on AVCRI104P3 as a promising multitarget therapeutic agent for the treatment of cholinergic dysfunction underlying natural aging and/or dementias. Copyright © 2015. Published by Elsevier B.V.

  8. Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

    Science.gov (United States)

    Shin, Areum; Lee, Eunjung; Jeon, Dasom; Park, Young-Guen; Bang, Jeong Kyu; Park, Yong-Sun; Shin, Song Yub; Kim, Yangmee

    2015-06-30

    Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

  9. Influence of Cell Detachment on the Respiration Rate of Tumor and Endothelial Cells

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F.; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study. PMID:23382841

  10. Influence of cell detachment on the respiration rate of tumor and endothelial cells.

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

  11. B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

    Science.gov (United States)

    Nakaya, K; Mizuno, R; Ohhashi, T

    2001-12-01

    We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

  12. Anti-Ma-associated encephalitis due to dysgerminoma in a woman with Swyer syndrome.

    Science.gov (United States)

    Al-Thubaiti, Ibtisam; Al-Hayek, Kefah; Binfalah, Mohammed

    2013-04-09

    Paraneoplastic limbic encephalitis (PLE)/diencephalitis associated with anti-Ma2 antibodies was linked to testicular cancer and non-small-cell lung cancer (non-SCLC).(1,2) We report a case of anti-Ma-associated PLE/diencephalitis due to dysgerminoma in a woman with gonadal dysgenesis, or Swyer syndrome.

  13. Human intestinal absorption of imidacloprid with Caco-2 cells as enterocyte model

    International Nuclear Information System (INIS)

    Brunet, Jean-Luc; Maresca, Marc; Fantini, Jacques; Belzunces, Luc P.

    2004-01-01

    In order to assess the risk to mammals of a chronic exposure to imidacloprid (IMI), we investigated its absorption with the human intestinal Caco-2 cell line. Measurements of transepithelial transport revealed an apparent permeability coefficient of 21.6 x 10 -6 ± 3.2 x 10 -6 cm/s reflecting a 100% absorption. The comparison of apical to basal (A-B) and basal to apical (B-A) transports showed that the monolayer presents a basal to apical polarized transport. Studies of apical uptake demonstrated that the transport was concentration-dependent and not saturable from 5 to 200 μM. Arrhenius plot analysis revealed two apparent activation energies, E a(4-12deg . C) = 63.8 kJ/mol and E a(12-37deg. C) 18.2 kJ/mol, suggesting two temperature-dependent processes. IMI uptake was equivalent when it was performed at pH 6.0 or 7.4. Depletion of Na + from the transport buffer did not affect the uptake, indicating that a sodium-dependent transporter was not involved. Decrease of uptake with sodium-azide or after cell surface trypsin (Ti) treatment suggested the involvement of a trypsin-sensitive ATP-dependent transporter. Investigations on apical efflux demonstrated that initial velocities paralleled the increase of loading concentrations. A cell surface trypsin treatment did not affect the apical efflux. The lack of effect when the efflux was performed against an IMI concentration gradient suggested that an energy-dependent transporter was involved. However, the inhibition of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRP) by taxol, vincristine, and daunorubicine had no effect on IMI intracellular accumulation suggesting the involvement of transporters distinct from classical ATP binding cassette transport (ABC-transport) systems. All results suggest that IMI is strongly absorbed in vivo by inward and outward active transporters

  14. BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells.

    Science.gov (United States)

    Dia, Vermont P; Krishnan, Hari B

    2016-09-15

    Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity of BG-4 may be responsible for its capability to cause cytotoxicity to HCT-116 and HT-29 human colon cancer cells with ED50 values of 134.4 and 217.0 μg/mL after 48 h of treatment, respectively. The mechanism involved in the cytotoxic effect may be associated with induction of apoptosis as evidenced by increased percentage of HCT-116 and HT-29 colon cancer cells undergoing apoptosis from 5.4% (untreated) to 24.8% (BG-4 treated, 125 μg/mL for 16 h) and 8.5% (untreated) to 31.9% (BG-4 treated, 125 μg/mL for 16 h), respectively. The molecular mechanistic explanation in the apoptosis inducing property of BG-4 is due to reduced expression of Bcl-2 and increased expression of Bax leading to increased expression of caspase-3 and affecting the expression of cell cycle proteins p21 and CDK2. This is the first report on the anti-cancer potential of a novel bioactive peptide isolated from Momordica charantia in vitro supporting the potential therapeutic property of BG-4 against colon cancer that must be addressed using in vivo models of colon carcinogenesis.

  15. Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells.

    Science.gov (United States)

    de Siqueira Patriota, Leydianne Leite; Procópio, Thamara Figueiredo; de Santana Brito, Jéssica; Sebag, Virginie; de Oliveira, Ana Patrícia Silva; de Araújo Soares, Ana Karine; Moreira, Leyllane Rafael; de Albuquerque Lima, Thâmarah; Soares, Tatiana; da Silva, Túlio Diego; Paiva, Patrícia Maria Guedes; de Lorena, Virgínia Maria Barros; de Melo, Cristiane Moutinho Lagos; de Albuquerque, Lidiane Pereira; Napoleão, Thiago Henrique

    2017-10-01

    In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25μg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5μg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8 + cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Scaling of X pinches from 1 MA to 6 MA

    International Nuclear Information System (INIS)

    Bland, Simon Nicholas; McBride, Ryan D.; Wenger, David Franklin; Sinars, Daniel Brian; Chittenden, Jeremy Paul; Pikuz, Sergei A.; Harding, Eric; Jennings, Christopher A.; Ampleford, David J.; Yu, Edmund P.; Cuneo, Michael Edward; Shelkovenko, Tatiana A.; Hansen, Stephanie B.

    2010-01-01

    This final report for Project 117863 summarizes progress made toward understanding how X-pinch load designs scale to high currents. The X-pinch load geometry was conceived in 1982 as a method to study the formation and properties of bright x-ray spots in z-pinch plasmas. X-pinch plasmas driven by 0.2 MA currents were found to have source sizes of 1 micron, temperatures >1 keV, lifetimes of 10-100 ps, and densities >0.1 times solid density. These conditions are believed to result from the direct magnetic compression of matter. Physical models that capture the behavior of 0.2 MA X pinches predict more extreme parameters at currents >1 MA. This project developed load designs for up to 6 MA on the SATURN facility and attempted to measure the resulting plasma parameters. Source sizes of 5-8 microns were observed in some cases along with evidence for high temperatures (several keV) and short time durations (<500 ps).

  17. Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

    International Nuclear Information System (INIS)

    Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

    2005-01-01

    Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as

  18. Effects of bleomycin and irradiation on euoxic and hypoxic cells

    International Nuclear Information System (INIS)

    Shrieve, D.C.; Harris, J.W.

    1979-01-01

    EMT6 cells in vitro were exposed to bleomycin (BLM), either alone (under euoxic or hypoxic conditions) or in conjunction with x-radiation. Hypoxic and euoxic cells were equally sensitive to the drug in both of the systems used to induce hypoxia (ampules or chambers). Exposure to BLM immediately before x-irradiation altered the shape of the radiation survival curve decreasing the D 0 by a factor of 1.3. Simultaneous exposure to x-ray and BLM resulted in lower survivals than when radiation was given either before or after drug treatment. Cells recovered quickly from BLM damage if trypsinization was delayed. The results indicate that BLM and x-rays interact to lower cell survival but that cells recover from this effect if trypsinization is delayed

  19. The Ste12-like transcription factor MaSte12 is involved in pathogenicity by regulating the appressorium formation in the entomopathogenic fungus, Metarhizium acridum.

    Science.gov (United States)

    Wei, Qinglv; Du, Yanru; Jin, Kai; Xia, Yuxian

    2017-12-01

    Homeodomain transcription factor Ste12 is a key target activated by the pathogenic mitogen-activated-protein kinase pathway, and the activated Ste12p protein regulates downstream gene expression levels to modulate phenotypes. However, the functions of Ste12-like genes in entomopathogenic fungi remain poorly understood and little is known about the downstream genes regulated by Ste12. In this study, we characterized the functions of a Ste12 orthologue in Metarhizium acridum, MaSte12, and identified its downstream target genes. The deletion mutant (ΔMaSte12) is defective in conidial germination but not in hyphal growth, conidiation, or stress tolerance. Bioassays showed that ΔMaSte12 had a dramatically decreased virulence in topical inoculations, but no significant difference was found in intrahemolymph injections when the penetration process was bypassed. The mature appressorium formation rate of ΔMaSte12 was less than 10% on locust wings, with the majority hyphae forming appressorium-like, curved but no swollen structures. Digital gene expression profiling revealed that some genes involved in cell wall synthesis and remodeling, appressorium development, and insect cuticle penetration were downregulated in ΔMaSte12. Thus, MaSte12 has critical roles in the pathogenicity of the entomopathogenic fungus M. acridum, and our study provides some explanations for the impairment of fungal virulence in ΔMaSte12. In addition, virulence is very important for fungal biocontrol agents to control insect pests effectively. This study demonstrated that MaSte12 is involved in fungal virulence but not conidial yield or fungal stress tolerance in M. acridum. Thus, MaSte12 and its downstream genes may be candidates for enhancing fungal virulence to improve mycoinsecticides.

  20. 5 CFR 2638.104 - Definitions.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Definitions. 2638.104 Section 2638.104 Administrative Personnel OFFICE OF GOVERNMENT ETHICS GOVERNMENT ETHICS OFFICE OF GOVERNMENT ETHICS AND EXECUTIVE AGENCY ETHICS PROGRAM RESPONSIBILITIES General Provisions § 2638.104 Definitions. For the purposes of...

  1. SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment

    Directory of Open Access Journals (Sweden)

    Genevieve V. Dall

    2017-02-01

    Full Text Available Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα. These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1, we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO, we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

  2. 28 CFR 551.104 - Housing.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Housing. 551.104 Section 551.104 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MISCELLANEOUS Pretrial Inmates § 551.104 Housing. To the extent practicable, pretrial inmates will be housed separately from convicted...

  3. Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death

    Directory of Open Access Journals (Sweden)

    Hou-Long Long

    2018-05-01

    Full Text Available Dietary anthocyanin compounds have multiple biological effects, including antioxidant, anti-inflammatory, and anti-atherosclerotic characteristics. The present study evaluated the anti-tumor capacity of mulberry anthocyanins (MA in thyroid cancer cells. Our data showed that MA suppressed SW1736 and HTh-7 cell proliferation in a time- and dose-dependent manner. Meanwhile, flow cytometry results indicated that MA significantly increased SW1736 and HTh-7 cell apoptosis. We additionally observed that SW1736 and HTh-7 cell autophagy was markedly enhanced after MA treatment. Importantly, anthocyanin-induced cell death was largely abolished by 3-methyladenine (3-MA or chloroquine diphosphate salt (CQ treatment, suggesting that MA-induced SW1736 and HTh-7 cell death was partially dependent on autophagy. In addition, activation of protein kinase B (Akt, mammalian target of rapamycin (mTOR, and ribosomal protein S6 (S6 were significantly suppressed by anthocyanin exposure. In summary, MA may serve as an adjunctive therapy for thyroid cancer patients through induction of apoptosis and autophagy-dependent cell death. Keywords: Mulberry anthocyanins, Thyroid cancer, Apoptosis, Autophagic death

  4. Discovery and utilization of sorghum genes (Ma5/Ma6)

    Science.gov (United States)

    Mullet, John E; Rooney, William L; Klein, Patricia E; Morishige, Daryl; Murphy, Rebecca; Brady, Jeff A

    2012-11-13

    Methods and composition for the production of non-flowering or late flowering sorghum hybrid. For example, in certain aspects methods for use of molecular markers that constitute the Ma5/Ma6 pathway to modulate photoperiod sensitivity are described. The invention allows the production of plants having improved productivity and biomass generation.

  5. Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells.

    Directory of Open Access Journals (Sweden)

    Islam Husain

    Full Text Available Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7-8 and temperature 35-40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10-3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h and trypsin (T1/2 = ~ 32 min half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1, which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.

  6. Effects of acylation on the functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate.

    Science.gov (United States)

    Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan

    2009-01-01

    The effects of succinylation and acetylation on some functional properties and the in vitro trypsin digestibility of kidney bean protein isolate (KPI) were investigated. The extent of succinylation or acetylation progressively increased from 0% to 96% to 97%, as the anhydride-to-protein ratio increased from 0 to 1 g/g. Polyacrylamide gel electrophoresis (PAGE) and zeta potential analyses indicated that acylation, especially succinylation, considerably increased the net charge and hydrodynamic radius of the proteins in KPI, especially vicilin. Acylation treatment at various anhydride-to-protein ratios (0.05 to 1 g/g) remarkably improved the protein solubility (PS) and emulsifying activity index (EAI) at neutral pH, but the improvement by succinylation was much better than that by acetylation. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependent manner. The results suggest that the functional properties of KPI could be modulated by the chemical acylation treatment, using succinic or acetic anhydride at appropriate anhydride-to-protein ratios.

  7. 34 CFR 104.45 - Housing.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Housing. 104.45 Section 104.45 Education Regulations of... Postsecondary Education § 104.45 Housing. (a) Housing provided by the recipient. A recipient that provides housing to its nonhandicapped students shall provide comparable, convenient, and accessible housing to...

  8. Biochemical response of Anticarsia gemmatalis fed with soybean plants pulverized with the synthetic trypsin inhibitor benzamidine

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, M.G.A.; Pilon, A.M.; Pilon, F.M.; Ribeiro, F.R.; Silva, F.C.; Ribon, A.O.B.; Reis, A.P.; Visotto, L.E. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Biologia Molecular; Guedes, R.N.C. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Biologia Animal; Oliveira, J.A. [Universidade Federal de Vicosa (UFV), Belo Horizonte, MG (Brazil). Dept. de Quimica

    2008-07-01

    Full text: Insects are responsible for severe crop losses. New alternatives for pest control other than agrochemicals have been investigated. Protease inhibitors are one of the prime candidates effective against insect pests. In this work we studied the effect of the synthetic trypsin inhibitor benzamidine on the development of Anticarsia gemmatalis, an important pest of the soybean culture. Larvae were reared on soybean plants containing 0.00, 0.15, 0.30, 0.45, 0.60 and 0.75% (w/w) of benzamidine. After 6, 12, 24 and 48 h of feeding midgut extracts were prepared and assayed for enzymatic activity (proteolytic, amidasic and stearic). Benzamidine altered the activity patterns but was not able to totally abolish enzyme activity. The proteolytic, amidasic and stearic activity showed the higher time of inhibition in 48 h in concentration of 0,75%, the inhibition was the around 93%, 63.1% and 36.6%, respectively. We suggest that the presence of inhibitor has made insects to adapt and produce proteases which are insensitive to the action of benzamidine. (author)

  9. Biochemical response of Anticarsia gemmatalis fed with soybean plants pulverized with the synthetic trypsin inhibitor benzamidine

    International Nuclear Information System (INIS)

    Oliveira, M.G.A.; Pilon, A.M.; Pilon, F.M.; Ribeiro, F.R.; Silva, F.C.; Ribon, A.O.B.; Reis, A.P.; Visotto, L.E.; Guedes, R.N.C.; Oliveira, J.A.

    2008-01-01

    Full text: Insects are responsible for severe crop losses. New alternatives for pest control other than agrochemicals have been investigated. Protease inhibitors are one of the prime candidates effective against insect pests. In this work we studied the effect of the synthetic trypsin inhibitor benzamidine on the development of Anticarsia gemmatalis, an important pest of the soybean culture. Larvae were reared on soybean plants containing 0.00, 0.15, 0.30, 0.45, 0.60 and 0.75% (w/w) of benzamidine. After 6, 12, 24 and 48 h of feeding midgut extracts were prepared and assayed for enzymatic activity (proteolytic, amidasic and stearic). Benzamidine altered the activity patterns but was not able to totally abolish enzyme activity. The proteolytic, amidasic and stearic activity showed the higher time of inhibition in 48 h in concentration of 0,75%, the inhibition was the around 93%, 63.1% and 36.6%, respectively. We suggest that the presence of inhibitor has made insects to adapt and produce proteases which are insensitive to the action of benzamidine. (author)

  10. Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

    Science.gov (United States)

    Riemann, Anne; Güttler, Antje; Haupt, Verena; Wichmann, Henri; Reime, Sarah; Bache, Matthias; Vordermark, Dirk; Thews, Oliver

    2018-03-05

    Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

  11. 32 CFR 643.104 - Consideration.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Consideration. 643.104 Section 643.104 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE Permits § 643.104 Consideration. (a) Permits are usually granted on a rent-free basis. (b) The Army is...

  12. 29 CFR 4022.104 - Examples.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Examples. 4022.104 Section 4022.104 Labor Regulations... Future Periods After Death § 4022.104 Examples. The following examples show how the rules in §§ 4022.101.... (1) Example 1: where surviving beneficiary predeceases participant. Ellen died before Charlie. As...

  13. Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Roh, Changhyun; Seo, Su-Hyun; Choi, Kwon-Young; Cha, Minho; Pandey, Bishnu Prasad; Kim, June-Hyung; Park, Jun-Seong; Kim, Duck Hee; Chang, Ih Seop; Kim, Byung-Gee

    2009-07-01

    Screening of bacterial whole cells was performed for regioselective hydroxylation of daidzein and genistein. Among the strains examined, Streptomyces avermitilis MA-4680 showed high ortho-dihydroxylation activity to produce 3',4',7-trihydroxyisoflavone and 3',4',5,7-tetrahydroxyisoflavone from daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone), respectively. Using 100 mg cells (wet wt.) and 1% (v/v) Triton X100 in 1 ml of total reaction volume, where 100 microl of the substrate solution (0.5 mM in 10% (v/v) mixed solvent of DMSO:MeOH = 3:7) was added to 900 microl of potassium phosphate buffer (100 mM, pH 7.2), a 16% molar conversion yield of 3',4',7-trihydroxyisoflavone was obtained from 0.5 mM daidzein after 24 h of reaction time at 28 degrees C and 200 rpm. Ketoconazole significantly (ca. 90%) inhibited the ortho-hydroxylation activity of daidzein, suggesting that cytochrome P450 enzymes putatively play roles in regiospecific daidzein hydroxylation. The analysis of the reaction products was determined by gas chromatography/mass spectrometry (GC/MS) and (1)H NMR.

  14. Differential stress response of Saccharomyces hybrids revealed by monitoring Hsp104 aggregation and disaggregation.

    Science.gov (United States)

    Kempf, Claudia; Lengeler, Klaus; Wendland, Jürgen

    2017-07-01

    Proteotoxic stress may occur upon exposure of yeast cells to different stress conditions. The induction of stress response mechanisms is important for cells to adapt to changes in the environment and ensure survival. For example, during exposure to elevated temperatures the expression of heat shock proteins such as Hsp104 is induced in yeast. Hsp104 extracts misfolded proteins from aggregates to promote their refolding. We used an Hsp104-GFP reporter to analyze the stress profiles of Saccharomyces species hybrids. To this end a haploid S. cerevisiae strain, harboring a chromosomal HSP104-GFP under control of its endogenous promoter, was mated with stable haploids of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum. Stress response behaviors in these hybrids were followed over time by monitoring the appearance and dissolution of Hsp104-GFP foci upon heat shock. General stress tolerance of these hybrids was related to the growth rate detected during exposure to e.g. ethanol and oxidizing agents. We observed that hybrids were generally more resistant to high temperature and ethanol stress compared to their parental strains. Amongst the hybrids differential responses regarding the appearance of Hsp104-foci and the time required for dissolving these aggregates were observed. The S. cerevisiae/S. paradoxus hybrid, combining the two most closely related strains, performed best under these conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. New advances in cell physiology and pathophysiology of the exocrine pancreas.

    Science.gov (United States)

    Mössner, Joachim

    2010-01-01

    This review provides some aspects on the physiology of stimulation and inhibition of pancreatic digestive enzyme secretion and the pathophysiology of pancreatic acinar cell function leading to pancreatitis. Cholecystokinin (CCK) stimulates both directly via CCK-A receptors on acinar cells and indirectly via CCK-B receptors on nerves, followed by acetylcholine release, pancreatic enzyme secretion. It is still not known whether CCK-A receptors exist in human acinar cells, in contrast to acinar cells of rodents where CCK-A receptors have been well described. CCK has numerous actions both in the periphery and in the central nervous systems. CCK inhibits gastric motility and regulates satiety. Another major function of CCK is stimulation of gallbladder contraction. This function enables that bile acids act simultaneously with pancreatic lipolytic enzymes. Secretin is a major stimulator of bicarbonate secretion. Trypsinogen is activated by the gut mucosal enzyme enterokinase. The other pancreatic proenzymes are activated by trypsin. Termination of enzyme secretion may be regulated by negative feedback mechanisms via destruction of CCK-releasing peptides by trypsin. Furthermore, the ileum may act as a brake by release of inhibitory hormones such as PYY and somatostatin. In the pathophysiology of acute pancreatitis, fusion of zymogen granules with lysosomes leading to intracellular activation of trypsinogen is regarded as an initiation step. This activation of trypsinogen may be caused by the lysosomal enzyme cathepsin B. However, autoactivation of trypsinogen itself may be a possibility in pathogenesis. Autoactivation is enhanced in certain mutations of trypsinogen. Furthermore, an imbalance of protease inhibitors and active proteases may be involved. The role of pancreatic lipolytic enzymes, the role of bicarbonate secretion, and toxic Ca(2+) signals by excessive liberation from the endoplasmic reticulum have to be discussed in the pathogenesis of acute pancreatitis

  16. Differential modulation of binding loop flexibility and stability by Arg50 and Arg52 in Cucurbita maxima trypsin inhibitor-V deduced by trypsin-catalyzed hydrolysis and NMR spectroscopy.

    Science.gov (United States)

    Cai, M; Huang, Y; Prakash, O; Wen, L; Dunkelbarger, S P; Huang, J K; Liu, J; Krishnamoorthi, R

    1996-04-16

    The side chains of Arg50 and Arg52 iin Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J.L-F., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys44-Asp45 peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CmTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on a reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate that Arg50 contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg50 and Arg52 were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: 15N NMR longitudinal and transverse relaxation rates and 15N-1H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in 15N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-59; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58 and 66 are found to be highly mobile, as the caluculated general order parameters, S2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg50 amd Arg52 are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are

  17. Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells.

    Science.gov (United States)

    Karlsson, Mattias; Scherbak, Nikolai; Khalaf, Hazem; Olsson, Per-Erik; Jass, Jana

    2012-11-01

    Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. [Comparative analysis of sensitivity of proteases (chymotrypsin and trypsin) and cholinesterases of different origin to some organophosphorus inhibitors].

    Science.gov (United States)

    Rozengart, E V

    2009-01-01

    The antichymotrypsin, antitrypsin, and anticholinesterase efficiencies of four homologous series of organophosphorus inhibitors are compared: O-ethyl-S-(n-alkyl)methylthiophosphonates, O-(n-alkyl)-S-(n-butyl)methylthiophosphonates, O-(n-alkyl)-S-beta-(ethylmercaptoethylene)methylthiophosphonates, and their methylsulfomethylates. As sources of a-chymotrypsin and trypsin, commercial compounds of Worthington Biochemical Corporation and Leningrad Myasokombinat were tested. Bimolecular constant of the reaction rate was used as the measure of antienzyme efficiency. In all cases, the antichymotrypsin efficiency was lower, while the antitrypsin--essentially higher than the anticholinesterase activity of the studied inhibitors. These differences were found to much depend both on the inhibitor structure and on nature of the cholinesterase compounds.

  19. Protective effects against H2O2-induced damage by enzymatic hydrolysates of an edible brown seaweed, sea tangle (Laminaria japonica).

    Science.gov (United States)

    Park, Pyo-Jam; Kim, Eun-Kyung; Lee, Seung-Jae; Park, Sun-Young; Kang, Dong-Soo; Jung, Bok-Mi; Kim, Kui-Shik; Je, Jae-Young; Ahn, Chang-Bum

    2009-02-01

    Enzymatic hydrolysates of Laminaria japonica were evaluated for antioxidative activities using hydroxyl radical scavenging activity and protective effects against H(2)O(2)-induced DNA and cell damage. In addition, activities of antioxidative enzymes, including catalase, glutathione peroxidase, and glutathione S-transferase, of the enzymatic hydrolysates from L. japonica were also estimated. L. japonica was first enzymatically hydrolyzed by seven carbohydrases (Dextrozyme, AMG, Promozyme, Maltogenase, Termamyl, Viscozyme, and Celluclast [all from Novo Co., Novozyme Nordisk, Bagsvaerd, Denmark]) and five proteinases (Flavourzyme, Neutrase, Protamex, Alcalase [all from Novo Co.], and pancreatic trypsin). The hydroxyl radical scavenging activities of Promozyme and pancreatic trypsin hydrolysates from L. japonica were the highest as compared to those of the other carbohydrases and proteinases, and their 50% inhibitory concentration values were 1.67 and 317.49 mug/mL, respectively. The pancreatic trypsin hydrolysates of L. japonica exerted a protective effect on H(2)O(2)-induced DNA damage. We also evaluated the protective effect on hydroxyl radical-induced oxidative damage in PC12 cells via propidium iodide staining using a flow cytometer. The AMG and pancreatic trypsin hydrolysates of L. japonica dose-dependently protected PC12 cells against cell death caused by hydroxyl radical-induced oxidative damage. Additionally, we analyzed the activity of antioxidative enzymes such as catalase, glutathione peroxidase, and the phase II biotransformation enzyme glutathione S-transferase in L. japonica-treated cells. The activity of all antioxidative enzymes was higher in L. japonica-treated cells compared with the nontreated cells. These results indicate that enzymatic hydrolysates of L. japonica possess antioxidative activity.

  20. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    International Nuclear Information System (INIS)

    Hovi, T.; Roivainen, M.

    1989-01-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with 51 Cr, [ 3 H]leucine, or, preferentially, with [ 3 H]uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30 degree C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well

  1. Immunoreactive trypsin and neonatalscreening for cystic fibrosis

    International Nuclear Information System (INIS)

    Travert, G.; Laroche, D.; Blandin, C.; Pasquet, C.

    1988-01-01

    Immunoreactive trypsin (IRT) was measured in dried blood spots from 160.822 five-day-old babies as a part of a regionwide neonatal screening program for cystic fibrosis. A second test was performed for 492 babies in whom blood IRT levels were found greater than 900 μg/l; retesting revealed persistent elevation in 55. Sweat testing confirmed cystic fibrosis in 43 babies, but results were normal in 12. During the course of this study, a total of 51 cystic fibrosis babies were identified: 43 by newborn screening, 6 because they had meconium ileus; so, early diagnosis was achieved in 49 cases out of 51. Two newborn babies did not have elevated IRT and they were missed by the screening test. Our results confirm that elevated blood IRT is characteristic of newborn babies with cystic fibrosis and show that this test has an excellent specificity (99.7%) and a good sensitivity (95%) when used as a neonatal screening test [fr

  2. A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

    International Nuclear Information System (INIS)

    Dao, T.; Holan, V.; Minowada, J.

    1993-01-01

    A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

  3. Electronic structure of trypsin inhibitor from squash seeds in aqueous solution

    Science.gov (United States)

    Zheng, Haoping

    2000-10-01

    The electronic structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima (CMTI-I) in aqueous solution is obtained by ab initio, all-electron, full-potential calculations using the self-consistent cluster-embedding (SCCE) method. The reactive site of the inhibitor is explained theoretically, which is in agreement with the experimental results. It is shown that the coordinates of oxygen atoms in the inhibitor, determined by nuclear magnetic resonance and combination of distance geometry and dynamical simulated annealing, are systematically less accurate than that of other kinds of heavy atoms.

  4. AMIODARONE INDUCES THE SYNTHESIS OF HSPS IN SACCHAROMYCES CEREVISIAE AND ARABIDOPSIS THALIANA CELLS

    Directory of Open Access Journals (Sweden)

    Pyatrikas D.V.

    2012-08-01

    Full Text Available Many biotic and abiotic stresses cause an increase of cytosolic Ca2+ level in cells. Calcium is one of the most important second messengers, regulating many various activities in the cell and was known to affect expression of stress activated genes. Mild heat shock induces the expression of heat shock proteins (Hsps which protect cell from drastic heat shock exposure. There are some literature data permitting to suggest that transient elevation of cytosolic Ca2+ level in plant cells is important for activation of Hsps expression. On the other hand mitochondria are known to regulate the intracellular calcium and reactive oxygen species signaling. It has been shown recently that mild heat shock induces hyperpolarization of inner mitochondrial membrane in plant and yeast cells and this event is critically important for activation of Hsps expression. To reveal the relationship between mitochondrial activity, intracellular calcium homeostasis and Hsps expression an antiarrhythmic drug amiodarone (AMD have been used. AMD is known to cause transient increase of cytosolic Ca2+ level in Saccharomyces cerevisiae. Obtained results have showed that AMD treatment induced the synthesis of Hsp104p in S. cerevisiae cells and Hsp101p in A. thaliana cell culture. Induction of Hsp104p synthesis leads to enhanced yeast capability to survive lethal heat shock exposure. Development of S. cerevisiae thermotolerance depended significantly on the presence of Hsp104p. Elevation of Hsp104p level in the result of AMD treatment was shown to be governed by activity of Msn2p and Msn4p transcription factors. Deletion of the MSN2 and MSN4 genes abrogated the AMD ability to induce Hsp104p synthesis. Mild heat shock and AMD treatment induced the hyperpolarization of the inner mitochondrial membrane in yeast and Arabidopsis cells which accompanied by HSP synthesis and development of thermotolerance. It was suggested that increase of cytosolic Ca2+ level after AMD treatment

  5. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

    Science.gov (United States)

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  6. Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

    Directory of Open Access Journals (Sweden)

    Selene Glück

    Full Text Available In infected cells rotavirus (RV replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2 involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.

  7. Faculty of Law: MaRBLe for Lawyers

    NARCIS (Netherlands)

    Akkermans, Bram; Bastiaens, Ellen; van Tilburg, Jonathan; van Merriënboer, Jeroen

    The Maastricht University Research Based Learning (MaRBLe) project offered a unique but challenging opportunity to the law faculty. Working with undergraduate (or bachelor) students on research was already part of regular, i.e. curricular, teaching activities, but the number of students and

  8. Structural and functional aspects of trypsin–gold nanoparticle interactions: An experimental investigation

    Energy Technology Data Exchange (ETDEWEB)

    Nidhin, Marimuthu [Department of Chemistry, Amity School of Applied Sciences, Center for Nanoscience and Technology, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413 (India); Ghosh, Debasree [Department of Nanotechnology, Amity School of Applied Sciences, Center for Nanoscience and Technology, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413 (India); Yadav, Himanshu; Yadav, Nitu [Department of Chemistry, Amity School of Applied Sciences, Center for Nanoscience and Technology, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413 (India); Majumder, Sudip, E-mail: sudip22m@gmail.com [Department of Chemistry, Amity School of Applied Sciences, Center for Nanoscience and Technology, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413 (India)

    2015-12-15

    Highlights: • Trypsin undergoes activation on incubation with gold nanoparticles. • Enhanced activity depends on the stoichiometry of the mixture. • Higher concentration of nanoparticles damage stability and conformation of trypsin. • Gold nanoparticles undergo morphological change on incubation with trypsin. - Abstract: Trypsin (Trp) is arguably the most important member of the serine proteases. Constructs made up of gold nanoparticles (GNP) with trypsin have been known to exhibit increased efficiency and stability in various experiments. Here we report simple Trp–GNP constructs mixed in different trypsin-to-GNP ratios which exhibit higher efficiencies in biochemical assay, varying resistance to autolysis and higher ability in cell trypsinization. Trp–GNP constructs in different trypsin-to-GNP ratios exhibit prolonged and sustained activity compared to native trypsin in N-α-p-benzoyl-p-nitroanilide (BAPNA) assay as monitored by UV-Visible spectroscopy. The activity was monitored as a function of decreasing rate of linear release of p-nitro aniline (resulting from the cleavage of BAPNA by trypsin) with time during the assay, whose absorbance was measured at 410 nm (λ{sub max} p-nitro aniline). We have done extensive studies to understand structural basis of this trypsin GNP interaction by using atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) techniques. Our findings suggest that on interaction, the gold nanoparticles probably form an adherent layer on trypsin that effectively changes the morphology and dimensions of the nanoconstructs. However, trypsin-to-GNP ratio is extremely important, as higher concentration of GNP might damage the conformation of protein. Stability studies related to denaturation show that 1:1 Trp–GNP constructs exhibit maximum stability and high efficiency in all assays performed.

  9. Structural and functional aspects of trypsin–gold nanoparticle interactions: An experimental investigation

    International Nuclear Information System (INIS)

    Nidhin, Marimuthu; Ghosh, Debasree; Yadav, Himanshu; Yadav, Nitu; Majumder, Sudip

    2015-01-01

    Highlights: • Trypsin undergoes activation on incubation with gold nanoparticles. • Enhanced activity depends on the stoichiometry of the mixture. • Higher concentration of nanoparticles damage stability and conformation of trypsin. • Gold nanoparticles undergo morphological change on incubation with trypsin. - Abstract: Trypsin (Trp) is arguably the most important member of the serine proteases. Constructs made up of gold nanoparticles (GNP) with trypsin have been known to exhibit increased efficiency and stability in various experiments. Here we report simple Trp–GNP constructs mixed in different trypsin-to-GNP ratios which exhibit higher efficiencies in biochemical assay, varying resistance to autolysis and higher ability in cell trypsinization. Trp–GNP constructs in different trypsin-to-GNP ratios exhibit prolonged and sustained activity compared to native trypsin in N-α-p-benzoyl-p-nitroanilide (BAPNA) assay as monitored by UV-Visible spectroscopy. The activity was monitored as a function of decreasing rate of linear release of p-nitro aniline (resulting from the cleavage of BAPNA by trypsin) with time during the assay, whose absorbance was measured at 410 nm (λ_m_a_x p-nitro aniline). We have done extensive studies to understand structural basis of this trypsin GNP interaction by using atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) techniques. Our findings suggest that on interaction, the gold nanoparticles probably form an adherent layer on trypsin that effectively changes the morphology and dimensions of the nanoconstructs. However, trypsin-to-GNP ratio is extremely important, as higher concentration of GNP might damage the conformation of protein. Stability studies related to denaturation show that 1:1 Trp–GNP constructs exhibit maximum stability and high efficiency in all assays performed.

  10. Morotochoerus from Uganda (17.5 Ma and Kenyapotamus from Kenya (13-11 Ma: implications for hippopotamid origins

    Directory of Open Access Journals (Sweden)

    Pickford, M.

    2011-12-01

    Full Text Available The aim of this paper is to describe and interpret suiform teeth from Moroto, Uganda, and Ngorora, Kenya, which contribute to the debate about hippo-anthracothere-whale relationships. The early stages of hippopotamid evolution are relatively poorly known on account of the paucity of their fossil record older than 7 Ma. New specimens of Morotochoerus from Uganda reveal that it is not closely related to Hippopotamidae; the superficial resemblances of the cheek teeth to those of hippos represent convergences and not homologies. Restricted samples of Palaeopotamus ternani are available from the Middle Miocene of Kenya {Maboko, ca 16 Ma; Muruyur, ca 14.5 Ma; Fort Ternan, ca 13.7 Ma} while from the base of the late Miocene, Kenyapotamus coryndonae is known from Kenya {Ngerngerwa, ca 10.5-10 Ma; Nakali, ca 10.5 Ma; Samburu Hills, ca 9.5 Ma}, Ethiopia {Ch’orora, ca 10.5 Ma} and Tunisia {Beglia Formation ca 11-10 Ma}. The recovery of specimens of Kenyapotamus from the Ngorora Formation, Kenya, aged ca 11 Ma, is of interest because it includes well preserved teeth, including an m/3 in good condition. These specimens support the hypothesis that hippopotamids descended from palaeochoerids and not from anthracotheres.El objetivo de este trabajo es describir e interpretar los dientes suiformes de Moroto, Uganda, y Ngorora, Kenia, que contribuyen al debate sobre las relaciones hipo-anthracothere-whale. Las primeras etapas de la evolución de los hipopotámidos son relativamente poco conocidas a causa de la escasez de su registro fósil en edades superiors a los 7 Ma. Nuevos ejemplares de Morotochoerus en Uganda revelan que no están estrechamente relacionados con Hippopotamidae, las semejanzas superficiales de los dientes de la mandíbula con los de los hipopótamos representan convergencias y no homologías. Algunas muestras de Palaeopotamus ternani aparecen en el Medio Mioceno de Kenia {Maboko, ca 16 Ma; Muruyur, ca 14.5 Ma; Fort Ternan, ca 13.7 Ma

  11. PC/104 Embedded IOCs at Jefferson Lab

    International Nuclear Information System (INIS)

    Yan, Jianxun; Allison, Trent; Witherspoon, Sue; Cuffe, Anthony

    2009-01-01

    Jefferson Lab has developed embedded IOCs based on PC/104 single board computers (SBC) for low level control systems. The PC/104 IOCs run EPICS on top of the RTEMS operating system. Two types of control system configurations are used in different applications, PC/104 SBC with commercial PC/104 I/O cards and PC/104 SBC with custom designed FPGA-based boards. RTEMS was built with CEXP shell to run on the PC/104 SBC. CEXP shell provides the function of dynamic object loading, which is similar to the widely used VxWorks operating system. Standard software configurations were setup for PC/104 IOC application development to provide a familiar format for new projects as well as ease the conversion of applications from VME based IOCs to PC/104 IOCs. Many new projects at Jefferson Lab are going to employ PC/104 SBCs as IOCs and some applications have already been running them for accelerator operations. The PC/104 - RTEMS IOC provides a free open source Real-Time Operating System (RTOS), low cost/maintenance, easily installed/ configured, flexible, and reliable solution for accelerator control and 12GeV Upgrade projects.

  12. Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues† †Electronic supplementary information (ESI) available: Synthesis of TEG–BA, Gluen–BA, mGluen–BA and Gluen–Ph; 1H NMR, 13C NMR, MALDI-TOF MS, electronic absorption, and CD spectra; zeta potential distributions; SLS plots; DLS histograms; and related experimental procedures. See DOI: 10.1039/c5sc00524h Click here for additional data file.

    Science.gov (United States)

    Mogaki, Rina

    2015-01-01

    Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Gluen–BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Gluen–BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10–BA and Glue29–BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG–BA). Most importantly, Glue10–BA inhibited the protease activity of trypsin 13-fold more than TEG–BA. In sharp contrast, mGlue27–BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG–BA for trypsin, was inferior even to TEG–BA for trypsin inhibition. PMID:28706668

  13. Dioscorin protects tight junction protein expression in A549 human airway epithelium cells from dust mite damage.

    Science.gov (United States)

    Fu, Lin Shien; Ko, Ying Hsien; Lin, Kuo Wei; Hsu, Jeng Yuan; Chu, Jao Jia; Chi, Chin Shiang

    2009-12-01

    In addition to being an allergen, the trypsin activity of dust mite extract also destroys the tight junctions of bronchial epithelium. Such damage can lead to airway leakage, which increases airway exposure to allergens, irritants, and other pathogens. Dioscorin, the storage protein of yam, demonstrates anti-trypsin activity, as well as other potential anti-inflammatory effects. This study investigated the protective role of dioscorin for tight junctions. The immunofluorescence stains of zonula occludens (ZO-1), E-cadherin (EC) and desmoplakin (DP) proteins were compared. A cultured A549 cell line was used as a control and A549 cells were incubated with mite extract 100 mg/mL for 16 h, with or without dioscorin 100 mg/mL pretreatment for 8 h and with dioscorin 100 mg/mL alone for 16 h. Western blot was performed to detect changes in ZO-1, EC, and DP in the treated A549 cell lines. Loss of tight junction protein expression (ZO-1, EC, DP) was demonstrated after 16-h mite extract incubation. The defect could be restored if cells were pretreated with dioscorin for 8 h. In addition, dioscorin did not cause damage to the A549 cell lines in terms of cell survival or morphology. Western blot showed no change in the amount of tight junction protein under various conditions. Dioscorin is a potential protector of airway damage caused by mite extract.

  14. 48 CFR 425.104 - Nonavailable articles.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Nonavailable articles. 425.104 Section 425.104 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE SOCIOECONOMIC PROGRAMS FOREIGN ACQUISITION Buy American Act-Supplies 425.104 Nonavailable articles. Information required...

  15. 11 CFR 104.10 - Reporting by separate segregated funds and nonconnected committees of expenses allocated among...

    Science.gov (United States)

    2010-01-01

    ...-Federal candidates. The committee shall also report the amount of each in-kind contribution, independent... nonconnected committees of expenses allocated among candidates and activities. 104.10 Section 104.10 Federal... among candidates and activities. (a) Expenses allocated among candidates. A political committee that is...

  16. Nitrite-reductase and peroxynitrite isomerization activities of Methanosarcina acetivorans protoglobin.

    Directory of Open Access Journals (Sweden)

    Paolo Ascenzi

    Full Text Available Within the globin superfamily, protoglobins (Pgb belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb, since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb* are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II is biphasic and values of the second-order rate constant for the reduction of NO2- to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II (Ma-Pgb*-Fe(II-NO are k(app1= 9.6 ± 0.2 M(-1 s(-1 and k(app2 = 1.2 ± 0.1 M(-1 s(-1 (at pH 7.4 and 20 °C. The k(app1 and k(app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h(app = 3.8 × 10(4 M(-1 s(-1 and h0 = 2.8 × 10(-1 s(-1 (at pH 7.4 and 20 °C. The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20 °C, indicating that HOONO is the species that reacts preferentially with the heme-Fe(III atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.

  17. Functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate: Effect of high-pressure treatment.

    Science.gov (United States)

    Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan; Li, Lin

    2008-10-15

    The effects of high-pressure (HP) treatment at 200-600MPa, prior to freeze-drying, on some functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. Surface hydrophobicity and free sulfhydryl (SH) and disulfide bond (SS) contents were also evaluated. HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in fluorescence strength and SS formation from SH groups, and decrease in denaturation enthalpy change. The protein solubility of KPI was significantly improved at pressures of 400MPa or higher, possibly due to formation of soluble aggregate from insoluble precipitate. HP treatment at 200 and 400MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI was significantly decreased at 600MPa (relative to untreated KPI). The thermal stability of the vicilin component was not affected by HP treatment. Additionally, in vitro trypsin digestibility of KPI was decreased only at a pressure above 200MPa and for long incubation time (e.g., 120min). The data suggest that some physiochemical and functional properties of vicilin-rich kidney proteins can be improved by means of high-pressure treatment. Copyright © 2008 Elsevier Ltd. All rights reserved.

  18. 34 CFR 104.42 - Admissions and recruitment.

    Science.gov (United States)

    2010-07-01

    ... predictor of success in the education program or activity in question and (ii) alternate tests or criteria... recipient is taking remedial action to correct the effects of past discrimination pursuant to § 104.6(a) or... of paragraph (b)(2) of this section, a recipient may base prediction equations on first year grades...

  19. Formation and action of oxygen activated species in cell cultures

    International Nuclear Information System (INIS)

    Hoffmann, M.E.; Meneghini, R.

    1982-01-01

    The differences of hydrogen peroxide sensibility of mammal cell lineages (man, mouse, chinese hamster) in culture are studied. The cellular survival and the frequency of DNA induced breaks by hydrogen peroxide are analysed. The efficiency of elimination of DNA breaks by cells is determined. The possible relation between the cell capacity of repair and its survival to hydrogen peroxide action is also discussed. (M.A.) [pt

  20. 2-Triazenoazaindoles: α novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells.

    Science.gov (United States)

    Kreutzer, Jan N; Salvador, Alessia; Diana, Patrizia; Cirrincione, Girolamo; Vedaldi, Daniela; Litchfield, David W; Issinger, Olaf-Georg; Guerra, Barbara

    2012-04-01

    Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases.

  1. 2-Triazenoazaindoles: A novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells

    Science.gov (United States)

    KREUTZER, JAN N.; SALVADOR, ALESSIA; DIANA, PATRIZIA; CIRRINCIONE, GIROLAMO; VEDALDI, DANIELA; LITCHFIELD, DAVID W.; ISSINGER, OLAF-GEORG; GUERRA, BARBARA

    2012-01-01

    Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases. PMID:22134789

  2. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Solberg, H; Løber, D; Eriksen, J

    1992-01-01

    -blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45......,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M...... to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen...

  3. Synthesis, biodistribution and in vitro evaluation of brain permeable high affinity type 2 cannabinoid receptor agonists [11C]MA2 and [18F]MA3

    Directory of Open Access Journals (Sweden)

    Muneer Ahamed

    2016-09-01

    Full Text Available Abstract The type 2 cannabinoid receptor (CB2 is a member of the endocannabinoid system and is known for its important role in (neuroinflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2 and a fluorine-18 ([18F]MA3 labeled analogue of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM. MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analogue 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  4. Model for Analysis of the Energy Demand (MAED) users' manual for version MAED-1

    International Nuclear Information System (INIS)

    1986-09-01

    This manual is organized in two major parts. The first part includes eight main sections describing how to use the MAED-1 computer program and the second one consists of five appendices giving some additional information about the program. Concerning the main sections of the manual, Section 1 gives a summary description and some background information about the MAED-1 model. Section 2 extends the description of the MAED-1 model in more detail. Section 3 introduces some concepts, mainly related to the computer requirements imposed by the program, that are used throughout this document. Sections 4 to 7 describe how to execute each of the various programs (or modules) of the MAED-1 package. The description for each module shows the user how to prepare the control and data cards needed to execute the module and how to interpret the printed output produced. Section 8 recapitulates about the use of MAED-1 for carrying out energy and electricity planning studies, describes the several phases normally involved in this type of study and provides the user with practical hints about the most important aspects that need to be verified at each phase while executing the various MAED modules

  5. 47 CFR 19.735-104 - Delegations.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Delegations. 19.735-104 Section 19.735-104 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMPLOYEE RESPONSIBILITIES AND CONDUCT General Provisions § 19.735-104 Delegations. (a) The Commission has delegated to the Chairman responsibility for the...

  6. 34 CFR 104.4 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Discrimination prohibited. 104.4 Section 104.4... ASSISTANCE General Provisions § 104.4 Discrimination prohibited. (a) General. No qualified handicapped person... otherwise be subjected to discrimination under any program or activitiy which receives Federal financial...

  7. The [Mo₆Cl14]2- Cluster is Biologically Secure and Has Anti-Rotavirus Activity In Vitro.

    Science.gov (United States)

    Rojas-Mancilla, Edgardo; Oyarce, Alexis; Verdugo, Viviana; Morales-Verdejo, Cesar; Echeverria, Cesar; Velásquez, Felipe; Chnaiderman, Jonas; Valiente-Echeverría, Fernando; Ramirez-Tagle, Rodrigo

    2017-07-05

    The molybdenum cluster [Mo₆Cl 14 ] 2- is a fluorescent component with potential for use in cell labelling and pharmacology. Biological safety and antiviral properties of the cluster are as yet unknown. Here, we show the effect of acute exposition of human cells and red blood cells to the molybdenum cluster and its interaction with proteins and antiviral activity in vitro. We measured cell viability of HepG2 and EA.hy926 cell lines exposed to increasing concentrations of the cluster (0.1 to 250 µM), by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Hemolysis and morphological alterations of red blood cells, obtained from healthy donors, exposed to the cluster (10 to 200 µM) at 37 °C were analyzed. Furthermore, quenching of tryptophan residues of albumin was performed. Finally, plaque formation by rotavirus SA11 in MA104 cells treated with the cluster (100 to 300 µM) were analyzed. We found that all doses of the cluster showed similar cell viability, hemolysis, and morphology values, compared to control. Quenching of tryptophan residues of albumin suggests a protein-cluster complex formation. Finally, the cluster showed antiviral activity at 300 µM. These results indicate that the cluster [Mo₆Cl 14 ] 2- could be intravenously administered in animals at therapeutic doses for further in vivo studies and might be studied as an antiviral agent.

  8. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.

    Science.gov (United States)

    Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Minami, Ryunosuke; Nakagoshi, Hideki; Adachi-Yamada, Takashi

    2014-12-20

    In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate. The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform Mud(PBD) and the two newly characterized isoforms Mud(L) and Mud(S) regulated them differently: Mud(L) repressed cell rounding, Mud(PBD) and Mud(S) oriented the spindle along the apico-basal axis, and Mud(S) and Mud(L) repressed central spindle assembly. Importantly, overexpression of Mud(S) induced binucleation even in standard proliferating cells such as those in imaginal discs. We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate

  9. Effect of irradiation and germination on trypsin inhibitor and protein content of chickpea (Cicer arietinum L.)

    International Nuclear Information System (INIS)

    Sattar, A.; Atta, S.; Akhtar, M.A.

    1990-01-01

    Effect of irradiation (0.005-0.20 kGy) and subsequent germination on trypsin inhibitor activity (TIA) and protein content of chickpea, was studied. The results revealed a significant linear relation (r = -0.981 to -0.992) between the loss of TIA and germination time, and the rate of TIA destruction increased with irradiation dose (p < 0.05). Maximum destruction (43.8%) of the TIA occurred on germination for 120 hr of 0.20 kGy sample (from 330.0 to 185.3 TIU/g). Initially protein content was 21.7% and the value significantly increased to maximum levels of 27.5% and 27.9% in distilled and tap water respectively during germination for 120 hr of 0.10 kGy sample (p < 0.05). Protein contents were not affected by irradiation

  10. Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites.

    Science.gov (United States)

    Okamura, Masashi; Yokoyama, Naoaki; Takabatake, Noriyuki; Okubo, Kazuhiro; Ikehara, Yuzuru; Igarashi, Ikuo

    2007-02-01

    In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.

  11. 23 CFR 500.104 - State option.

    Science.gov (United States)

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false State option. 500.104 Section 500.104 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION TRANSPORTATION INFRASTRUCTURE MANAGEMENT MANAGEMENT AND MONITORING SYSTEMS Management Systems § 500.104 State option. Except as specified in § 500.105 (a...

  12. 28 CFR 301.104 - Medical attention.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Medical attention. 301.104 Section 301.104 Judicial Administration FEDERAL PRISON INDUSTRIES, INC., DEPARTMENT OF JUSTICE INMATE ACCIDENT COMPENSATION General § 301.104 Medical attention. Whenever an inmate worker is injured while in the performance...

  13. 28 CFR 104.32 - Eligibility review.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Eligibility review. 104.32 Section 104.32 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) SEPTEMBER 11TH VICTIM COMPENSATION FUND OF 2001 Claim Intake, Assistance, and Review Procedures § 104.32 Eligibility review. Any claimant deemed...

  14. 21 CFR 814.104 - Original applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Original applications. 814.104 Section 814.104...) MEDICAL DEVICES PREMARKET APPROVAL OF MEDICAL DEVICES Humanitarian Use Devices § 814.104 Original... applicant. (d) Address for submissions and correspondence. Copies of all original HDEs amendments and...

  15. Urinary trypsin inhibitor - an experimental and clinical study

    International Nuclear Information System (INIS)

    Berling, B.M.

    1991-01-01

    The urinary trypsin inhibitor (UTI) is an acid stable proteinase inhibitor present in blood and urine. It was purified from urine using affinity chromatography, ion exchange chromatography and gel filtration. Two forms of UTI were present in urine, A and B. A radioimmunoassay for measurement of UTI in urine and plasma was performed. The normal level of UTI in plasma and serum was about 2 mg/l. The normal excretion in urine was about 8 mg per 24 hours. The plasma and urine levels of UTI were studied in patients with acute pancreatitis and in patients undergoing cholecystectomy. Uremic patients had a marked increase of UTI in plasma compatible with decreased glomerular filtration. In samples from healthy persons as well as from patients only inhibitor A was found. Inhibitor B has recently been renamed bikunin because of its two Kunitz-type inhibiting domains. Inhibitor A might be called tetrakunin. Radioactively labeled UTI (inhibitor A) was injected intravenously in three male volunteers. The plasma half-life of 125 I UTI was 2 hours. Free biologically active inhibitor was found in the urine during the first four hours after injection. The organ distribution of intravenously injected 125 I UTI was studied in rats. Fifteen minutes after injection the major part of the radioactivity was found in the kidneys, suggesting that the kidneys are the primary site of UTI metabolism. Using immunohistochemical techniques UTI was found in the proximal tubules of the normal human kidney further indicating the tubular reabsorption and methabolisms of UTI

  16. 34 CFR 104.39 - Private education.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Private education. 104.39 Section 104.39 Education Regulations of the Offices of the Department of Education OFFICE FOR CIVIL RIGHTS, DEPARTMENT OF EDUCATION... Preschool, Elementary, and Secondary Education § 104.39 Private education. (a) A recipient that provides...

  17. 17 CFR 201.104 - Business hours.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Business hours. 201.104 Section 201.104 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION RULES OF PRACTICE Rules of Practice General Rules § 201.104 Business hours. The Headquarters office of the Commission, at...

  18. 34 CFR 104.11 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Discrimination prohibited. 104.11 Section 104.11... ASSISTANCE Employment Practices § 104.11 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or...

  19. [Herbological studies on Chinese crude drug Ma-huang. Part 1-On the botanical origin of Ma-huang in ancient China and the origin of Japanese Ma-huang].

    Science.gov (United States)

    Yoshizawa, Chieko; Kitade, Makiko; Mikage, Masayuki

    2005-01-01

    The botanical origin of a Chinese crude drug Ma-huang in ancient China and the origin of Japanese Ma-huang were herbologically studied. The results showed that the plants of Ephedra sinica Stapf, E. intermedia Schrenk & C. A. Meyer and E. equisetina Bunge were used as Ma-huang in China, and the first species was considered to be of high quality. The characters of Mao-zhou Ma-huang and Tong-zhou Ma-huang printed in Tu-jing-ben-cao, published in the Song Dynasty in China, were identified as E. likiangensis Florin and E. intermedia, respectively, and both species were recognized as excellent Ma-huang in the Ming Dynasty. The word origin of Katsune-kusa, the Japanese name for Ma-huang in the Heian Era, was etymologically considered as meaning the plant having reddish brown roots. In Japan, the plant of Equisetum ramosissimum Desf. var.japonicum Milde, of the family Equisetaceae, was substituted for Ma-huang in the middle of Edo Era, and it was designated that this action was based on the confusion of Ephedra plants and Equisetum plants those days in China.

  20. O6-alkylguanine-DNA-alkyltransferase activity and nitrosourea sensitivity in human cancer cell lines.

    OpenAIRE

    Walker, M. C.; Masters, J. R.; Margison, G. P.

    1992-01-01

    The DNA repair enzyme, O6-alkylguanine-DNA-alkyltransferase (ATase), is thought to be the principal mechanism controlling resistance to nitrosoureas and related alkylating agents. We compared the sensitivities of five human testis and five bladder tumour cell lines to two nitrosoureas (N-nitroso-N-methylurea (MNU) and mitozolomide) with cellular levels of ATase. Enzyme levels ranged from 3 to 206 fmol mg-1 protein (0.1 x 10(4) to 5.1 x 10(4) molecules/cell) in the testis lines and from 11 to ...

  1. A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple.

    Science.gov (United States)

    Bai, Yang; Dougherty, Laura; Li, Mingjun; Fazio, Gennaro; Cheng, Lailiang; Xu, Kenong

    2012-08-01

    Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R (2) = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS(1455) that targets the SNP(1455) in Ma1 showed that the CAPS(1455A) allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma

  2. Effect of enzyme location on activity and stability of trypsin and urease immobilized on porous membranes by using layer-by-layer self-assembly of polyelectrolyte

    OpenAIRE

    Guedidi, Sadika; Yürekli, Yılmaz; Deratani, André; Déjardin, Philippe; Innocent, Christophe; Altınkaya, Sacide; Roudesli, Sadok; Yemenicioğlu, Ahmet

    2010-01-01

    The layer-by-layer (LbL) self-assembly of polyelectrolyte is one of the simplest ways to immobilize enzyme on membrane. In this paper, the immobilization of trypsin (TRY) and urease (URE) on polyacrylonitrile based membranes using the LbL assembly technique was presented. The studied systems consisted in bilayered assemblies with the enzyme layer as the outer layer and trilayered assemblies with the enzyme layer as the inner sandwiched layer. The membrane pore size was chosen so that the smal...

  3. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  4. 34 CFR 104.23 - New construction.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false New construction. 104.23 Section 104.23 Education... Accessibility § 104.23 New construction. (a) Design and construction. Each facility or part of a facility..., if the construction was commenced after the effective date of this part. (b) Alteration. Each...

  5. 21 CFR 104.5 - General principles.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 2 2010-04-01 2010-04-01 false General principles. 104.5 Section 104.5 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION NUTRITIONAL QUALITY GUIDELINES FOR FOODS General Provisions § 104.5 General principles. (a) A...

  6. Vietnam Inbound M&A Activity: The Role of Government Policy and Regulatory Environment

    Directory of Open Access Journals (Sweden)

    Jamshid C. Hosseini

    2017-05-01

    Full Text Available With a robust recent history of reform and opening, joining of the World Trade Organization, and negotiating a myriad of regional and global trade agreements, Vietnam has emerged as a promising destination for foreign direct investment(FDI and cross-border mergers and acquisitions (M&A. In this paper, we providean overview of Vietnam’s inbound mergers and acquisitions and review the twomain driving forces of inbound M&A, which are the legal framework reformprocess and the equitization of State-owned enterprises. We close by providingdirections for future research in the area of cross-border M&As.

  7. 7 CFR 1160.104 - United States.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true United States. 1160.104 Section 1160.104 Agriculture... Definitions § 1160.104 United States. United States means the 48 contiguous states in the continental United States and the District of Columbia, except that United States means the 50 states of the United States...

  8. 28 CFR 104.22 - Advance Benefits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Advance Benefits. 104.22 Section 104.22... Filing for Compensation; Application for Advance Benefits § 104.22 Advance Benefits. (a) Advance Benefits. Eligible Claimants may apply for immediate “Advance Benefits” in a fixed amount as follows: (1) $50,000 for...

  9. 48 CFR 48.104-3 - Sharing collateral savings.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Sharing collateral savings... CONTRACT MANAGEMENT VALUE ENGINEERING Policies and Procedures 48.104-3 Sharing collateral savings. (a) The Government shares collateral savings with the contractor, unless the head of the contracting activity has...

  10. Cell mediated therapeutics for cancer treatment: Tumor homing cells as therapeutic delivery vehicles

    Science.gov (United States)

    Balivada, Sivasai

    Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V

  11. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  12. Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

    Science.gov (United States)

    Ma, Xingzhe; Bi, Enguang; Huang, Chunjian; Lu, Yong; Xue, Gang; Guo, Xing; Wang, Aibo; Yang, Maojie; Qian, Jianfei; Dong, Chen; Yi, Qing

    2018-05-09

    CD8 + T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8 + or CD4 + T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function. © 2018 Ma et al.

  13. 5 CFR 536.104 - Reasonable offer.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Reasonable offer. 536.104 Section 536.104... Provisions § 536.104 Reasonable offer. (a) For the purpose of determining whether grade retention eligibility or entitlement must be terminated under § 536.207 or 536.208, the offer of a position is a reasonable...

  14. 7 CFR 25.104 - Poverty rate.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Poverty rate. 25.104 Section 25.104 Agriculture Office... § 25.104 Poverty rate. (a) General. Eligibility of an area on the basis of poverty shall be established in accordance with the following poverty rate criteria specific to Round I, Round II, Round IIS and...

  15. Enhancement of Immune Activation Activities of Spirulina maxima Grown in Deep-Sea Water

    Science.gov (United States)

    Choi, Woon Yong; Kang, Do Hyung; Lee, Hyeon Yong

    2013-01-01

    In this study, the immuno-modulatory and anticancer activities of marine algae, Spirulina maxima grown in deep-sea water (DSW), were investigated. It was found that the extract of S. maxima, cultured in DSW, effectively suppressed the expression of Bcl2 in A549 cells as well as inhibiting various human cancer cells with concentration dependency, which possibly implies that the extracts may play more important roles in controlling cancer cell growth. The secretion of cytokines IL-6 and TNF-α from human B cells was also greatly increased, compared to those of the extract grown in conventional sea-water. The growth of Human Natural Killer (NK) cells in the presence of the extracts from DSW was significantly higher (12.2 × 104 viable cells/mL) when compared to the control (1.1 × 104 viable cells/mL). Based on HPLC analysis, the increase in the biological activities of the extracts from DSW was caused by considerably high amounts of β-carotene and ascorbic acid because the DSW contained high concentrations and good ratios of several key minerals for biosynthesizing β-carotene and ascorbic acid, as well as maintaining high cell growth. PMID:23743830

  16. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

    Science.gov (United States)

    Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman

    2012-02-02

    Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Paraneoplastic brain stem encephalitis in a woman with anti-Ma2 antibody.

    Science.gov (United States)

    Barnett, M; Prosser, J; Sutton, I; Halmagyi, G M; Davies, L; Harper, C; Dalmau, J

    2001-02-01

    A woman developed brain stem encephalopathy in association with serum anti-Ma2 antibodies and left upper lobe lung mass. T2 weighted MRI of the brain showed abnormalities involving the pons, left middle and superior cerebellar peduncles, and bilateral basal ganglia. Immunohistochemical analysis for serum antineuronal antibodies was confounded by the presence of a non-neuronal specific antinuclear antibody. Immunoblot studies showed the presence of anti-Ma2 antibodies. A premortem tissue diagnosis of the lung mass could not be established despite two CT guided needle biopsies, and the patient died as a result of rapid neurological deterioration. The necropsy showed that the lung lesion was an adenocarcinoma which expressed Ma2 immunoreactive protein. Neuropathological findings included prominent perivascular inflammatory infiltrates, glial nodules, and neuronophagia involving the brain stem, basal ganglia, hippocampus and the dentate nucleus of the cerebellum. Ma2 is an autoantigen previously identified in patients with germ cell tumours of the testis and paraneoplastic brain stem and limbic encephalitis. Our patient's clinical and immunopathological findings indicate that this disorder can affect women with lung adenocarcinoma, and that the encephalitic changes predominate in those regions of the brain known to express high concentrations of Ma proteins.

  18. [IL-2 stimulated responses of CD3(+) CD56(+) NKT cells in pulmonary tuberculosis patients].

    Science.gov (United States)

    Yao, Chun-Yan; Wang, Zhao-Hua; Jiang, Li-Na; Peng, Mei-Yu; Wang, Jing; Li, Bai-Qing

    2010-09-01

    To observe the activation and proliferation characteristics of IL-2 stimulated CD3(+);CD56(+); NKT cells in pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells (PBMCs) from PTB patients and normal subjects were stimulated with IL-2 and cultured for different time points. The CD69 expression on and amount of the CD3(+);CD56(+); NKT cells were detected by multi fluorescence staining and flow cytometry at different time of stimulation and culture. There was no significant difference in percentage of NKT cells between PTB patients and normal healthy controls before culture. When IL-2 was used to stimulate for 0 h, 8 h, 16 h, 40 h and 64 h, the expression of CD69 on NKT cells in normal controls and PTB patients increased significantly, but the CD69 expression level of NKT cells in PTB patients was significantly higher than that in normal persons(PNKT cells increased from (3.44+/-1.20)x10(4); to (323.23+/-75.98) x10(4); (PNKT cells increased from (5.57+/-5.16)x10(4); to (1475.05+/-868.98)x10(4); (PNKT cells in PTB patients present with high activation but low proliferation after stimulated by IL-2.

  19. Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

    Science.gov (United States)

    Gardner, Qurra-tul-Ann Afza; Younas, Hooria; Akhtar, Muhammad

    2013-01-01

    Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

    Science.gov (United States)

    Taupin, P; Roisin, M P; Ben-Ari, Y; Barbin, G

    1994-06-27

    Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

  1. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

    Science.gov (United States)

    Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

    2017-01-01

    Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

  2. NMR characterisation of the minimal interacting regions of centrosomal proteins 4.1R and NuMA1: effect of phosphorylation

    Directory of Open Access Journals (Sweden)

    Bruix Marta

    2010-01-01

    Full Text Available Abstract Background Some functions of 4.1R in non-erythroid cells are directly related with its distinct sub-cellular localisation during cell cycle phases. During mitosis, 4.1R is implicated in cell cycle progression and spindle pole formation, and co-localizes with NuMA1. However, during interphase 4.1R is located in the nucleus and only partially co-localizes with NuMA1. Results We have characterized by NMR the structural features of the C-terminal domain of 4.1R and those of the minimal region (the last 64 residues involved in the interaction with NuMA1. This subdomain behaves as an intrinsically unfolded protein containing a central region with helical tendency. The specific residues implicated in the interaction with NuMA1 have been mapped by NMR titrations and involve the N-terminal and central helical regions. The segment of NuMA1 that interacts with 4.1R is phosphorylated during mitosis. Interestingly, NMR data indicates that the phosphorylation of NuMA1 interacting peptide provokes a change in the interaction mechanism. In this case, the recognition occurs through the central helical region as well as through the C-terminal region of the subdomain meanwhile the N-terminal region do not interact. Conclusions These changes in the interaction derived from the phosphorylation state of NuMA1 suggest that phosphorylation can act as subtle mechanism of temporal and spatial regulation of the complex 4.1R-NuMA1 and therefore of the processes where both proteins play a role.

  3. Basic Surface Properties of Mononuclear Cells from Didelphis marsupialis

    Directory of Open Access Journals (Sweden)

    Nacife Valéria Pereira

    1998-01-01

    Full Text Available The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals and -29.3 mV (cells from adult animals. The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5°and 40.8°, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

  4. Midgut Protease Activity During Larval Development of Anastrepha obliqua (Diptera: Tephritidae) Fed With Natural and Artificial Diet

    Science.gov (United States)

    Rivera-Ciprian, José Pedro; Aceituno-Medina, Marysol; Guillen, Karina

    2017-01-01

    Abstract In this study, we examined the activity of two serine proteases (chymotrypsin and trypsin) and two metalloproteases (carboxypeptidases A and B) during larval development in Anastrepha obliqua fed natural (mango fruit) and artificial (formulation used in mass-rearing) diets. Proteolytic activity of chymotrypsin, trypsin, carboxypeptidase A, and carboxypeptidase B was detected in the midgut of different instars of A. obliqua and was strongly affected by the pH and diet type. The protein content of the natural and artificial diets was similar. Enzymatic activity was higher in the midgut of the larvae fed the natural diet than in larvae fed the artificial diet. The activity of the endopeptidases (chymotrypsin and trypsin) was lower than those of the exopeptidases (carboxypeptidases A and B). The pH of the midgut varied from acidic to neutral. The results indicate that in the midgut of the larvae reared on both types of diet, the level of carboxypeptidase activity was approximately 100-fold greater than the level of chymotrypsin activity and 10,000-fold greater than the level of trypsin. In conclusion, carboxypeptidase A and B are the main proteases involved in the digestion of proteins in the larvae of A. obliqua. The natural diet showed a high bioaccessibility. A clear tendency to express high activities of chymotrypsin and trypsin was observed by the third instar. Our research contributes to the planning and development of novel bioaccessibility assays to understand the nutrition processing of A. obliqua larvae under mass-rearing conditions for sterile insect technique.

  5. Regulation of Banana Phytoene Synthase (MaPSY) Expression, Characterization and Their Modulation under Various Abiotic Stress Conditions

    Science.gov (United States)

    Kaur, Navneet; Pandey, Ashutosh; Shivani; Kumar, Prateek; Pandey, Pankaj; Kesarwani, Atul K.; Mantri, Shrikant S.; Awasthi, Praveen; Tiwari, Siddharth

    2017-01-01

    Phytoene synthase (PSY) is a key regulatory enzyme of carotenoid biosynthesis pathway in plants. The present study examines the role of PSY in carotenogenesis and stress management in banana. Germplasm screening of 10 Indian cultivars showed that Nendran (3011.94 μg/100 g dry weight) and Rasthali (105.35 μg/100 g dry weight) contained the highest and lowest amounts of β-carotene, respectively in ripe fruit-pulp. Nendran ripe pulp also showed significantly higher antioxidant activity as compared to Rasthali. Meta-analysis of three banana PSY genes (MaPSY1, MaPSY2, and MaPSY3) was performed to identify their structural features, subcellular, and chromosomal localization in banana genome. The distinct expression patterns of MaPSY1, MaPSY2, and MaPSY3 genes were observed in various tissues, and fruit developmental stages of these two contrasting cultivars, suggesting differential regulation of the banana PSY genes. A positive correlation was observed between the expression of MaPSY1 and β-carotene accumulation in the ripe fruit-peel and pulp of Nendran. The presence of stress responsive cis-regulatory motifs in promoter region of MaPSY genes were correlated with the expression pattern during various stress (abscisic acid, methyl jasmonate, salicylic acid and dark) treatments. The positive modulation of MaPSY1 noticed under abiotic stresses suggested its role in plant physiological functions and defense response. The amino acid sequence analysis of the PSY proteins in contrasting cultivars revealed that all PSY comprises conserved domains related to enzyme activity. Bacterial complementation assay has validated the functional activity of six PSY proteins and among them PSY1 of Nendran (Nen-PSY1) gave the highest activity. These data provide new insights into the regulation of PSY expression in banana by developmental and stress related signals that can be explored in the banana improvement programs. PMID:28421096

  6. Anti Ma2-associated myeloradiculopathy: expanding the phenotype of anti-Ma2 associated paraneoplastic syndromes

    OpenAIRE

    Murphy, Sinead M; Khan, Usman; Alifrangis, Constantine; Hazell, Steven; Hrouda, David; Blake, Julian; Ball, Joanna; Gabriel, Carolyn; Markarian, Pierre; Rees, Jeremy; Karim, Abid; Seckl, Michael J; Lunn, Michael P; Reilly, Mary M

    2011-01-01

    Anti-Ma2 associated paraneoplastic syndrome usually presents as limbic encephalitis in association with testicular tumours.1, 2 Only four patients have been reported with involvement outside the CNS, two of whom also had limbic or brainstem encephalitis.2, 3 We report a man with anti- Ma2 associated myeloradiculopathy and previous testicular cancer whose neurological syndrome stabilised and anti-Ma2 titres fell following orchidectomy of a microscopically normal testis.

  7. THE DISTANCE TO M104

    Energy Technology Data Exchange (ETDEWEB)

    McQuinn, Kristen B. W. [University of Texas at Austin, McDonald Observatory, 2515 Speedway, Stop C1400 Austin, TX 78712 (United States); Skillman, Evan D. [Minnesota Institute for Astrophysics, School of Physics and Astronomy, 116 Church Street, SE, University of Minnesota, Minneapolis, MN 55455 (United States); Dolphin, Andrew E. [Raytheon Company, 1151 E. Hermans Road, Tucson, AZ 85756 (United States); Berg, Danielle [Center for Gravitation, Cosmology and Astrophysics, Department of Physics, University of Wisconsin Milwaukee, 1900 East Kenwood Boulevard, Milwaukee, WI 53211 (United States); Kennicutt, Robert, E-mail: kmcquinn@astro.as.utexas.edu [Institute for Astronomy, University of Cambridge, Madingley Road, Cambridge CB3 0HA (United Kingdom)

    2016-11-01

    M104 (NGC 4594; the Sombrero galaxy) is a nearby, well-studied elliptical galaxy included in scores of surveys focused on understanding the details of galaxy evolution. Despite the importance of observations of M104, a consensus distance has not yet been established. Here, we use newly obtained Hubble Space Telescope optical imaging to measure the distance to M104 based on the tip of the red giant branch (TRGB) method. Our measurement yields the distance to M104 to be 9.55 ± 0.13 ± 0.31 Mpc equivalent to a distance modulus of 29.90 ± 0.03 ± 0.07 mag. Our distance is an improvement over previous results as we use a well-calibrated, stable distance indicator, precision photometry in a optimally selected field of view, and a Bayesian maximum likelihood technique that reduces measurement uncertainties. The most discrepant previous results are due to Tully–Fisher method distances, which are likely inappropriate for M104 given its peculiar morphology and structure. Our results are part of a larger program to measure accurate distances to a sample of well-known spiral galaxies (including M51, M74, and M63) using the TRGB method.

  8. Organizational behavior of human umbilical vein endothelial cells

    Science.gov (United States)

    1982-01-01

    Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization. PMID:6813338

  9. Patent Analysis for Supporting Merger and Acquisition (M&A) Prediction: A Data Mining Approach

    Science.gov (United States)

    Wei, Chih-Ping; Jiang, Yu-Syun; Yang, Chin-Sheng

    M&A plays an increasingly important role in the contemporary business environment. Companies usually conduct M&A to pursue complementarity from other companies for preserving and/or extending their competitive advantages. For the given bidder company, a critical first step to the success of M&A activities is the appropriate selection of target companies. However, existing studies on M&A prediction incur several limitations, such as the exclusion of technological variables in M&A prediction models and the omission of the profile of the respective bidder company and its compatibility with candidate target companies. In response to these limitations, we propose an M&A prediction technique which not only encompasses technological variables derived from patent analysis as prediction indictors but also takes into account the profiles of both bidder and candidate target companies when building an M&A prediction model. We collect a set of real-world M&A cases to evaluate the proposed technique. The evaluation results are encouraging and will serve as a basis for future studies.

  10. 48 CFR 32.104 - Providing contract financing.

    Science.gov (United States)

    2010-10-01

    ... financing. 32.104 Section 32.104 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT FINANCING Non-Commercial Item Purchase Financing 32.104 Providing contract financing. (a) Prudent contract financing can be a useful working tool in Government...

  11. The role of proteases, endoplasmic reticulum stress and SERPINA1 heterozygosity in lung disease and alpha-1 anti-trypsin deficiency.

    LENUS (Irish Health Repository)

    Greene, Catherine M

    2012-02-01

    The serine proteinase inhibitor alpha-1 anti-trypsin (AAT) provides an antiprotease protective screen throughout the body. Mutations in the AAT gene (SERPINA1) that lead to deficiency in AAT are associated with chronic obstructive pulmonary diseases. The Z mutation encodes a misfolded variant of AAT that is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum of hepatocytes and other AAT-producing cells. Until recently, it was thought that loss of antiprotease function was the major cause of ZAAT-related lung disease. However, the contribution of gain-of-function effects is now being recognized. Here we describe how both loss- and gain-of-function effects can contribute to ZAAT-related lung disease. In addition, we explore how SERPINA1 heterozygosity could contribute to smoking-induced chronic obstructive pulmonary diseases and consider the consequences.

  12. 36 CFR 406.104-406.109 - [Reserved

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false [Reserved] 406.104-406.109 Section 406.104-406.109 Parks, Forests, and Public Property AMERICAN BATTLE MONUMENTS COMMISSION... BATTLE MONUMENTS COMMISSION §§ 406.104-406.109 [Reserved] ...

  13. Production of Xylanase by Recombinant Bacillus subtilis DB104 Cultivated in Agroindustrial Waste Medium

    Directory of Open Access Journals (Sweden)

    Is Helianti

    2016-07-01

    Full Text Available A recombinant Bacillus subtilis DB104 strain harbouring recombinant plasmid pSKE194 containing an Open Reading Frame (ORF of endoxylanase and its indigenous promoter from the wild-type B. subtilis AQ1 strain was constructed. This recombinant B. subtilis DB104 strain had higher endoxylanase activity than the nonrecombinant B. subtilis DB104 strain in standard media, such as Luria Bertani (LB and LB with xylan. The agroindustrial wastes corncobs and tofu liquid waste were chosen as cost-effective carbon and nitrogen sources, respectively, to test the economics of xylanase production using the recombinant B. subtilis DB104 at a larger scale. Submerged fermentation using a 4.5 L working volume fermentor with tofu liquid waste and 4% corncobs produced maximum xylanase activity of 1296 ± 1.2 U/mg (601.7 ± 0.6 U/mL after 48-hour fermentation at 37°C with 150 rpm agitation; this is more than twofold higher than the activity produced in an Erlenmeyer flask. This is the first report of high xylanase activity produced from recombinant B. subtilis using inexpensive medium. During fermentation, the xylanase degrades corncobs into xylooligosaccharides, showing its potential as an enzyme feed additive or in xylooligosaccharide production.

  14. Tank 241-A-104 tank characterization plan

    International Nuclear Information System (INIS)

    Schreiber, R.D.

    1994-01-01

    This document is a plan which serves as the contractual agreement between the Characterization Program, Sampling Operations, WHC 222-S Laboratory, and PNL 325 Analytical Chemistry Laboratory. The scope of this plan is to provide guidance for the sampling and analysis of auger samples from tank 241-A-104. This Tank Characterization Plan will identify characterization objectives pertaining to sample collection, hot cell sample isolation, and laboratory analytical evaluation and reporting requirements in addition to reporting the current contents and status of the tank as projected from historical information

  15. Influence of relativistic effect on chemical properties of element 104; Wplyw efektu relatywistycznego na wlasnosci chemiczne pierwiastka 104

    Energy Technology Data Exchange (ETDEWEB)

    Bilewicz, A.

    1997-12-31

    The influence of relativistic effect upon chemical properties of element 104 is discussed. An original method of measurements of adsorption on the surface of thin film of cobalt ferrocyanate was developed and applied for the studies of 104{sup 4+} hydrolysis. Results of this experiments indicate that in the Group 4 tendency to hydrolysis decreases in the order 104{sup 4+}>Zr{sup 4+}>Hf{sup 4+}. The results were explained on the basis of relativistic effect. Unexpected chemical properties of element 104 in aqueous solutions indicate, that due to relativistic effect element 104 differs distinctly from its congeners - Zr and Hf. In contrary it becomes similar to the lightest element in the Group, Ti, through atomic mass of latter is 213 unit less. (author). 119 refs, 22 figs, 7 tabs.

  16. Supplementation with a new trypsin inhibitor from peanut is associated with reduced fasting glucose, weight control, and increased plasma CCK secretion in an animal model.

    Science.gov (United States)

    Serquiz, Alexandre C; Machado, Richele J A; Serquiz, Raphael P; Lima, Vanessa C O; de Carvalho, Fabiana Maria C; Carneiro, Marcella A A; Maciel, Bruna L L; Uchôa, Adriana F; Santos, Elizeu A; Morais, Ana H A

    2016-12-01

    Ingestion of peanuts may have a beneficial effect on weight control, possibly due to the satietogenic action of trypsin inhibitors. The aim of this study was to isolate a new trypsin inhibitor in a typical Brazilian peanut sweet (paçoca) and evaluate its effect in biochemical parameters, weight gain and food intake in male Wistar rats. The trypsin inhibitor in peanut paçoca (AHTI) was isolated. Experimental diets were prepared with AIN-93G supplemented with AHTI. Animals had their weight and food intake monitored. Animals were anesthetized, euthanized, and their bloods collected by cardiac puncture for dosage of cholecystokinin (CCK) and other biochemical parameters. Supplementation with AHTI significantly decreased fasting glucose, body weight gain, and food intake. These effects may be attributed to increased satiety, once supplemented animals showed no evidence of impaired nutritional status and also because AHTI increased CCK production. Thus, our results indicate that AHTI, besides reducing fasting glucose, can reduce weight gain via food intake reduction.

  17. Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    Directory of Open Access Journals (Sweden)

    Defeng Wu

    2013-01-01

    Full Text Available Autophagy has been shown to be protective against drug and alcohol-induced liver injury. CYP2E1 plays a role in the toxicity of ethanol, carcinogens and certain drugs. Inhibition of autophagy increased ethanol-toxicity and accumulation of fat in wild type and CYP2E1 knockin mice but not in CYP2E1 knockout mice as well as in HepG2 cells expressing CYP2E1 (E47 cells but not HepG2 cells lacking CYP2E1 (C34 cells. The goal of the current study was to evaluate whether modulation of autophagy can affect CYP2E1-dependent cytotoxicity in the E47 cells. The agents used to promote CYP2E1 –dependent toxicity were a polyunsaturated fatty acid, arachidonic acid (AA, buthionine sulfoximine (BSO, which depletes GSH, and CCl4, which is metabolized to the CCl3 radical. These three agents produced a decrease in E47 cell viability which was enhanced upon inhibition of autophagy by 3-methyladenine (3-MA or Atg 7 siRNA. Toxicity was lowered by rapamycin which increased autophagy and was much lower to the C34 cells which do not express CYP2E1. Toxicity was mainly necrotic and was associated with an increase in reactive oxygen production and oxidative stress; 3-MA increased while rapamycin blunted the oxidative stress. The enhanced toxicity and ROS formation produced when autophagy was inhibited was prevented by the antioxidant N-Acetyl cysteine. AA, BSO and CCl4 produced mitochondrial dysfunction, lowered cellular ATP levels and elevated mitochondrial production of ROS. This mitochondrial dysfunction was enhanced by inhibition of autophagy with 3-MA but decreased when autophagy was increased by rapamycin. The mitogen activated protein kinases p38 MAPK and JNK were activated by AA especially when autophagy was inhibited and chemical inhibitors of p38 MAPK and JNK lowered the elevated toxicity of AA produced by 3-MA. These results show that autophagy was protective against the toxicity produced by several agents known to be activated by CYP2E1. Since CYP2E1 plays an

  18. Radiation transformation in differentiated human cells in culture

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.; Moriarty, M.; Malone, J.; Byrne, P.; Hennessy, T.

    1986-01-01

    A tissue culture technique is described for human thyroid tissue as an approach to studying mechanisms of human radiation carcinogenesis. Normal human tissue obtained from surgery is treated in one of two ways, depending upon size of specimen. Large pieces are completely digested in trypsin/ collagenase solution to a single cell suspension. Small pieces of tissue are plated as explants following partial digestion in trypsin/collagenase solution. Following irradiation of the primary differentiated monolayers (normally 10 days after plating), the development of transformed characteristics is monitored in the subsequent subcultures. A very high level of morphological and functional differentiation is apparent in the primary cultures. Over a period of approx. 6 months, the irradiated surviving cells continue to grow in culture, unlike the unirradiated controls which senesce after 2-3 subcultures. (UK)

  19. Second primary tumor in anti-Ma1/2-positive paraneoplastic limbic encephalitis.

    Science.gov (United States)

    Leyhe, T; Schüle, R; Schwärzler, F; Gasser, T; Haarmeier, T

    2006-05-01

    Memory loss can be a symptom of paraneoplastic limbic encephalitis (PLE) a neuropsychiatric disorder associated mostly with small-cell lung cancer and anti-Hu antibodies or with testicular tumors and anti-Ma2 antibodies. We present the case of a patient with temporal coincidence of beginning cognitive decline and diagnosis of a carcinoma of the prostate in whom we diagnosed anti-Ma1/Ma2-positive PLE. The tumor had been completely resected but memory impairment further deteriorated. As the effective treatment of the cancer is considered as the most efficient treatment of a paraneoplastic neurological syndrome (PNS) a second neoplasia was suspected in the patient. By the aid of whole body positron emission tomography with 18-fluorine fluoro-2-deoxy-glucose (FDG-PET) an adenocarcinoma of the cecum could be detected. Two months after surgery anti-Ma antibodies were negative. We conclude that a second neoplasia should be considered, if effective cancer treatment does not lead to improvement or stabilisation of a PNS. Tumor search should be exhaustive and include PET when conventional imaging fails to show a malignancy.

  20. 7 CFR 987.104 - Major marketing promotion.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Major marketing promotion. 987.104 Section 987.104... IN RIVERSIDE COUNTY, CALIFORNIA Administrative Rules Definitions § 987.104 Major marketing promotion. A major marketing promotion program is one requiring the expenditure of more than $500 of Committee...

  1. 45 CFR 2490.104-2490.109 - [Reserved

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false [Reserved] 2490.104-2490.109 Section 2490.104-2490.109 Public Welfare Regulations Relating to Public Welfare (Continued) JAMES MADISON MEMORIAL... CONDUCTED BY THE JAMES MADISON MEMORIAL FELLOWSHIP FOUNDATION §§ 2490.104-2490.109 [Reserved] ...

  2. Asymmetrical squaraines for high-performance small-molecule organic solar cells with a short circuit current of over 12 mA cm(-2).

    Science.gov (United States)

    Chen, Yao; Zhu, Youqin; Yang, Daobin; Luo, Qian; Yang, Lin; Huang, Yan; Zhao, Suling; Lu, Zhiyun

    2015-04-11

    An asymmetrical squaraine dye (Py-3) with its two electron-donating aryl groups directly linked to the electron-withdrawing squaric acid core possesses an ideal bandgap of 1.33 eV, together with an intense and broad absorption band in the range 550-950 nm. Hence, the resulting solution-processed solar cells display an impressive Jsc of 12.03 mA cm(-2) and a PCE of 4.35%.

  3. Photothermal enhancement of chemotherapy mediated by gold-silica nanoshell-loaded macrophages: in vitro squamous cell carcinoma study

    Science.gov (United States)

    Madsen, Steen J.; Shih, En-Chung; Peng, Qian; Christie, Catherine; Krasieva, Tatiana; Hirschberg, Henry

    2016-01-01

    Moderate hyperthermia (MHT) has been shown to enhance the effects of chemotherapeutic agents in a wide variety of cancers. The purpose of this study was to investigate the combined effects of commonly used chemotherapeutic agents with MHT induced by near-infrared (NIR) activation of gold nanoshell (AuNS)-loaded macrophages (Ma). AuNS-loaded murine Ma combined with human FaDu squamous cells, in hybrid monolayers, were subjected to three cytotoxic drugs (doxorubicin, bleomycin, cisplatin) with or without NIR laser irradiation. For all three drugs, efficacy was increased by NIR activation of AuNS-loaded Ma. The results of this in vitro study provide proof-of-concept for the use of AuNS-loaded Ma for photothermal enhancement of the effects of chemotherapy on squamous cell carcinoma.

  4. Framework for Interpretation of Trypsin–antitrypsin Imbalance and Genetic Heterogeneity in Pancreatitis

    Science.gov (United States)

    Lin, Kun; Gao, Feng; Chen, Qingquan; Liu, Qicai; Chen, Shu

    2015-01-01

    Early intracellular premature trypsinogen activation was interpreted as the key initiator of pancreatitis. When the balance in the homeostasis of trypsin and antitrypsin system is disequilibrated, elevated aggressive enzymes directly attack the pancreatic tissue, which leads to pancreatic destruction and inflammation. However, trypsin alone is not enough to cause complications in pancreatitis, which may play a crucial role in modulating signaling events in the initial phase of the disease. NFκB activation is the major inflammatory pathway involved in the occurrence and development of pancreatitis and it can be induced by intrapancreatic activation of trypsinogen. Synthesis of trypsinogen occurs in endoplasmic reticulum (ER), and ER stress is an important early acinar cell event. Components of ER stress response are known to be able to trigger cell death as well as NFκB signaling cascade. The strongest evidence supporting the trypsin-centered theory is that gene mutations, which lead to the generation of more trypsin, or reduce the activity of trypsin inhibitors or trypsin degradation, are associated with pancreatitis. Thus, trypsin–antitrypsin imbalance may be the first step leading to pancreatic autodigestion and inducing other pathways. Continued experimental studies are necessary to determine the specific relationships between trypsin–antitrypsin imbalance and genetic heterogeneity in pancreatitis. In this article, we review the latest advances that contributed to the understanding of the basic mechanisms behind the occurrence and development of pancreatitis with a focus on the interpretation of trypsin–antitrypsin imbalance and their relationships with other inflammation pathways. We additionally highlight genetic predispositions to pancreatitis and possible mechanisms associated with them. PMID:26228362

  5. 19 CFR 358.104 - Report.

    Science.gov (United States)

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Report. 358.104 Section 358.104 Customs Duties... Report. The Secretary will review and issue a report on the first five years of the operation of Part 358. The report will consider the impact of determinations to permit importation of particular merchandise...

  6. Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

    Science.gov (United States)

    Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

    2013-09-01

    Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

    Science.gov (United States)

    Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

    1999-03-15

    We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the mo