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Sample records for active native trypsin

  1. The Activity of Trypsin

    ERIC Educational Resources Information Center

    Russo, Salvatore F.; Holzman, Tom

    1977-01-01

    Describes an experiment that illustrates the following points concerning the experimental determination of trypsin activity: (1) there is a difference in basing enzyme concentration on weight, absorbance, or active sites; and (2) the method of expressing enzyme concentration determines the value of specific, molecular, and catalytic center…

  2. Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation.

    PubMed

    Halangk, Walter; Krüger, Burkhard; Ruthenbürger, Manuel; Stürzebecher, Jörg; Albrecht, Elke; Lippert, Hans; Lerch, Markus M

    2002-02-01

    A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor Nalpha-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely prevented the generation of trypsin activity in response to supramaximal caerulein but had no effect whatsoever on the generation of TAP. Conversely, when trypsin activity was recovered at the end of the experiment by either washout of S124 from acini or extensive dilution of lobule homogenates, it was up to 400% higher than after caerulein alone and corresponded, in molar terms, to the generation of TAP. Both trypsin activity and TAP release were inhibited in parallel by E-64d. We conclude that caerulein-induced trypsinogen activation in the pancreas is caused by an E-64d-inhibitable mechanism such as cathepsin-induced trypsinogen activation, and neither involves nor requires intracellular trypsin activity. Specific trypsin inhibition, on the other hand, prevents 80% of trypsin inactivation or autodegradation in the pancreas.

  3. Synthesis of a cyanopeptide-analogue with trypsin activating properties.

    PubMed

    Radau, G; Rauh, D

    2000-04-17

    An efficient synthesis of a peptidic analogue of cyanobacterial metabolites with proposed serine protease inhibitory activity has been developed. Surprisingly, one trypsin activating compound was obtained.

  4. Proteolytic processing of native Cry1Ab toxin by midgut extracts and purified trypsins from the Mediterranean corn borer Sesamia nonagrioides.

    PubMed

    Díaz-Mendoza, Mercedes; Farinós, Gema P; Castañera, Pedro; Hernández-Crespo, Pedro; Ortego, Félix

    2007-05-01

    The proteolytic processing of native Cry1Ab toxin by midgut extracts from the Mediterranean corn borer, Sesamia nonagrioides, takes place in successive steps. Several cuts occur until a 74 kDa protein is obtained; this is further digested to give rise to an active form of 69 kDa, which can be again processed to fragments of 67, 66 and 43 kDa. We have shown that three different trypsins (TI, TIIA and TIII) purified from the S. nonagrioides midgut were able to digest Cry1Ab protoxin to obtain the active form of 69 kDa. Interestingly, TI and TIII further hydrolyzed the 69 kDa protein to a fragment of slightly lower molecular mass (67 kDa), while TIIA was able to continue digestion to give fragments of 46 and 43 kDa. These results contrast with those obtained using bovine trypsin, in which the main product of Cry1Ab digestion is a 69 kDa protein. The digestion of the toxin with a "non-trypsin" fraction from S. nonagrioides midgut lumen, mostly containing chymotrypsins and elastases and free of trypsin-like activity, resulted in a different processing pattern, yielding fragments of 79, 77, 71, 69 and 51 kDa. Our results indicate that trypsins and other proteases are involved in the first steps of protoxin processing, but trypsins play the most important role in obtaining the 74 and 69 kDa proteins. All the digestion products, including the proteins of 46 and 43 kDa obtained from the digestion of Cry1Ab by TIIA, were toxic to neonate larvae, indicating that none of the tested proteases contribute to toxin degradation in a significant manner.

  5. CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR

    PubMed Central

    Kunitz, M.

    1947-01-01

    A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496

  6. Crystal structure at 1.63 A resolution of the native form of porcine beta-trypsin: revealing an acetate ion binding site and functional water network.

    PubMed

    Johnson, A; Gautham, N; Pattabhi, V

    1999-11-16

    The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine beta-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 A resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-HellipsisO hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cbeta-Ogamma-His-57 Nepsilon angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.

  7. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

    NASA Astrophysics Data System (ADS)

    Luo, Wen; Zhao, Yunlong; Zhou, Zhongliang; An, Chuanguang; Ma, Qiang

    2008-02-01

    The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR (FQ—PCR) methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

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

    PubMed

    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.

  9. A trypsin inhibitor from snail medic seeds active against pest proteases.

    PubMed

    Ceciliani, F; Tava, A; Iori, R; Mortarino, M; Odoardi, M; Ronchi, S

    1997-02-01

    A protein trypsin inhibitor from seeds of snail medic (Medicago scutellata), MsTI, has been purified by ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin, with an apparent Kd of 1.8 x 10(-9), but exhibits no activity towards bovine alpha-chymotrypsin. Moreover, MsTI inhibits the trypsin-like proteinase activity present in larvae of the crop pests Adoxophyes orana, Hyphantria cunea, Lobesia botrana and Ostrinia nubilalis. The complete amino acid sequence of MsTI consists of 62 residues corresponding to a M(r) of 6925. Sequence comparison shows that MsTI exhibits significant similarity to other proteins belonging to the Bowman-Birk trypsin inhibitor family, and the closest identity (81%) with the wound-induced trypsin inhibitor from Medicago sativa leaves.

  10. Label-free, turn-on fluorescent sensor for trypsin activity assay and inhibitor screening.

    PubMed

    Zhang, Lufeng; Qin, Haiyan; Cui, Wanwan; Zhou, Yang; Du, Jianxiu

    2016-12-01

    The development of new detection methods for proteases activity assay is important in clinical diagnostics and drug development. In this work, a simple, label-free, and turn-on fluorescent sensor was fabricated for trypsin, a protease produced in the pancreas. Cytochrome c, a natural substance of trypsin, could be selectively cleaved by trypsin into heme-peptide fragment. The produced heme-peptide fragment exhibited an intensive catalytic role on the H2O2-mediated the oxidation of thiamine to form strong fluorescent thiochrome. The fluorescence intensity was closely dependent on the amount of trypsin presented. The procedure allowed the measurement of trypsin over the range of 0.5-20.0μg/mL with a detection limit of 0.125μg/mL. The sensor showed better precision with a relative standard deviation of 1.6% for the measurement of 1.0μg/mL trypsin solution (n=11). This sensing system was applied to screen the inhibitor of trypsin, the IC50 values were calculated to be 12.71ng/mL for the trypsin inhibitor from soybean and 2.0μg/mL for benzamidine hydrochloride, respectively, demonstrating its potential application in drug development and related diseases treatment.

  11. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity.

    PubMed

    Kaur, Gurvir; Tripathi, S K

    2015-01-05

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  12. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity

    NASA Astrophysics Data System (ADS)

    Kaur, Gurvir; Tripathi, S. K.

    2015-01-01

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  13. Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

    SciTech Connect

    Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

    2008-05-28

    Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineered Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.

  14. Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy

    PubMed Central

    Zhang, Yunfeng; Ling, Zhenmin; Du, Guocheng; Chen, Jian; Kang, Zhen

    2016-01-01

    N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml−1 (amidase activity, 8532 ± 142.2 U·ml−1 BAEE activity) with a productivity of 0.49 U·ml−1·h−1, which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes. PMID:26983398

  15. Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

    PubMed

    Lefkowitz, Roy B; Schmid-Schönbein, Geert W; Heller, Michael J

    2010-07-01

    The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

  16. The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum.

    PubMed Central

    Brock, J H; Arzabe, R; Piñeiro, A; Olivito, A M

    1977-01-01

    Digestion of bovine colostral whey with trypsin or chymotrypsin caused a progressive loss of the complement-mediated bactericidal activity of naturally-occurring colostral antibodies of E. coli 0111. Bactericidal activity was associated primarily with IgG1 immunoglobulin and to a lesser extent with IgM. Chymotrypsin preferentially attacked IgM, destroying its antibacterial activity and producing an apparent decrease in its mol wt. Trypsin preferentially attacked IgG1, but loss of antibacterial activity was in this case not accompanied by a decrease in molecular weight. Using colostral whey with antiperoxidase activity it was shown that the kinetics of loss of specific antibody activity were similar to those of loss of bactericidal activity. It is therefore suggested that trypsin may cause a loss of specific antibody activity of colostral IgG1 without cleaving the immunoglobulin molecule. PMID:321342

  17. Continuous method to determine the trypsin inhibitor activity in soybean flour.

    PubMed

    Coscueta, Ezequiel R; Pintado, Manuela E; Picó, Guillermo A; Knobel, Gastón; Boschetti, Carlos E; Malpiedi, Luciana Pellegrini; Nerli, Bibiana B

    2017-01-01

    The determination of trypsin inhibitor (TI) activity is of importance to evaluate the nutritional value of soybean flours. An analytical method, which involves a continuous spectrophotometric rate determination for trypsin activity against the substrate N-benzoyl-DL-arginine p-nitroanilide, is proposed as an alternative to the standard discontinuous assay. Stopping the reaction with acetic acid and a centrifugation/filtration step to decrease turbidity are not required, thus reducing costs and sample preparation time. The TI activity of different flour samples, determined by both assays, demonstrated to be statistically comparable, irrespective of the TI concentration level. The coefficients of variation of the novel method did not exceed 8% at any concentration level. The curves of progress reaction showed a non-linear behavior in samples without TI. A reduction of incubation time from 10min to 2min increased the method sensitivity and extended its linear range. A more economical, faster and simpler assay was developed.

  18. Development of activity-based probes for trypsin-family serine proteases.

    PubMed

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

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

    PubMed

    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.

  20. The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

    PubMed

    Perdomo-Morales, Rolando; Montero-Alejo, Vivian; Corzo, Gerardo; Besada, Vladimir; Vega-Hurtado, Yamile; González-González, Yamile; Perera, Erick; Porto-Verdecia, Marlene

    2013-11-01

    The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.

  1. The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

    PubMed Central

    Perdomo-Morales, Rolando; Montero-Alejo, Vivian; Corzo, Gerardo; Besada, Vladimir; Vega-Hurtado, Yamile; González-González, Yamile; Perera, Erick; Porto-Verdecia, Marlene

    2013-01-01

    The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme. PMID:24047891

  2. Potential Use of Atlantic Cod Trypsin in Biomedicine

    PubMed Central

    Gudmundsdóttir, Ágústa; Hilmarsson, Hilmar; Stefansson, Bjarki

    2013-01-01

    Surface proteins of viruses and bacteria used for cell attachment and invasion are candidates for degradation by proteases. Trypsin from Atlantic cod (Gadus morhua) was previously demonstrated to have efficacy against influenza viruses in vitro and on skin. In this paper, cod trypsin is shown to be 3–12 times more effective in degrading large native proteins than its mesophilic analogue, bovine trypsin. This is in agreement with previous findings where cod trypsin was found to be the most active among twelve different proteases in cleaving various cytokines and pathological proteins. Furthermore, our results show that cod trypsin has high efficacy against herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) in vitro. The results on the antipathogenic properties of cod trypsin are important because rhinovirus, RSV, and influenza are the most predominant pathogenic viruses in upper respiratory tract infections. Results from a clinical study presented in this paper show that a specific formulation containing cod trypsin was preferred for wound healing over other methods used in the study. Apparently, the high digestive ability of the cold-adapted cod trypsin towards large native proteins plays a role in its efficacy against pathogens and its positive effects on wounds. PMID:23555095

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

    PubMed Central

    Zhuo, Cai-Xia; Wang, Li-Hui; Feng, Jing-Jing; Zhang, Yao-Dong

    2016-01-01

    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. PMID:27834849

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

    PubMed

    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.

  5. Isolation and characterization of a Kunitz-type trypsin inhibitor with antiproliferative activity from Gymnocladus chinensis (Yunnan bean) seeds.

    PubMed

    Zhu, M J; Zhang, G Q; Wang, H X; Ng, T B

    2011-04-01

    A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC(50) value of 0.4 μM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-(3)H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC(50) value of 4.7 and 9.4 μM, respectively. There was no effect on human immunodeficiency virus(4)-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 μM.

  6. Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain.

    PubMed

    Lu, D; Yuan, X; Zheng, X; Sadler, J E

    1997-12-12

    Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean

  7. Effects of Croton rhamnifolioides essential oil on Aedes aegypti oviposition, larval toxicity and trypsin activity.

    PubMed

    Santos, Geanne K N; Dutra, Kamilla A; Lira, Camila S; Lima, Bheatriz N; Napoleão, Thiago H; Paiva, Patrícia M G; Maranhão, Claudia A; Brandão, Sofia S F; Navarro, Daniela M A F

    2014-10-14

    Although numerous reports are available concerning the larvicidal potential of essential oils, very few investigations have focused on their mechanisms of action. In the present study, we have investigated the chemical composition of the leaf oil of Croton rhamnifolioides during storage and its effects on oviposition and survival of larvae of the dengue fever mosquito Aedes aegypti. In addition, we have established a possible mechanism of action for the larvicidal activity of the essential oil. GC-MS analyses revealed marked differences in the composition of oil that had been freshly isolated and that of a sample that had been stored in a sealed amber-glass vial under refrigeration for three years. However, both fresh and stored oil exhibited substantial larvicidal activities with LC50 values of 122.35 and 89.03 ppm, respectively, and oviposition deterrent effects against gravid females at concentrations of 50 and 100 µg·mL-1. These results demonstrate that the larvicidal effect of the essential oil was unchanged during three years of storage even though its chemical composition altered. Hence, the essential oil could be used in the preparation of commercial products. In addition, we observed that the trypsin-like activity of mosquito larvae was inhibited in vitro by the essential oil of C. rhamnifolioides, suggesting that the larvicidal effect may be associated with inhibition of this enzyme.

  8. Antimicrobial Activity of ILTI, a Kunitz-Type Trypsin Inhibitor from Inga laurina (SW.) Willd.

    PubMed

    Macedo, Maria Lígia R; Ribeiro, Suzanna F F; Taveira, Gabriel B; Gomes, Valdirene M; de Barros, Karina M C A; Maria-Neto, Simone

    2016-05-01

    Over the last few years, a growing number of proteinase inhibitors have been isolated from plants and particularly from seeds and have shown antimicrobial activity. A 20,000 Da serine peptidase inhibitor, named ILTI, was isolated from Inga laurina seeds and showed potent inhibitory enzymatic activity against trypsin. The aim of this study was to determine the effects of ILTI on the growth of pathogenic and non-pathogenic microorganisms. We observed that ILTI strongly inhibited in particular the growth of Candida tropicalis and Candida buinensis, inducing cellular agglomeration. However, it was ineffective against human pathogenic bacteria. We also investigated the potential of ILTI to permeabilize the plasma membrane of yeast cells. C. tropicalis and C. buinensis were incubated for 24 h with the ILTI at different concentrations, which showed that this inhibitor induced changes in the membranes of yeast cells, leading to their permeabilization. Interestingly, ILTI induced the production of reactive oxygen species (ROS) in C. tropicalis and C. buinensis cells. Finally, ILTI was coupled with fluorescein isothiocyanate, and subsequent treatment of C. tropicalis and C. buinensis with DAPI revealed the presence of the labeled protein in the intracellular spaces. In conclusion, our results indicated the ability of peptidase inhibitors to induce microbial inhibition; therefore, they might offer templates for the design of new antifungal agents.

  9. Isothermal titration calorimetry determination of individual rate constants of trypsin catalytic activity.

    PubMed

    Aguirre, César; Condado-Morales, Itzel; Olguin, Luis F; Costas, Miguel

    2015-06-15

    Determination of individual rate constants for enzyme-catalyzed reactions is central to the understanding of their mechanism of action and is commonly obtained by stopped-flow kinetic experiments. However, most natural substrates either do not fluoresce/absorb or lack a significant change in their spectra while reacting and, therefore, are frequently chemically modified to render adequate molecules for their spectroscopic detection. Here, isothermal titration calorimetry (ITC) was used to obtain Michaelis-Menten plots for the trypsin-catalyzed hydrolysis of several substrates at different temperatures (278-318K): four spectrophotometrically blind lysine and arginine N-free esters, one N-substituted arginine ester, and one amide. A global fitting of these data provided the individual rate constants and activation energies for the acylation and deacylation reactions, and the ratio of the formation and dissociation rates of the enzyme-substrate complex, leading also to the corresponding free energies of activation. The results indicate that for lysine and arginine N-free esters deacylation is the rate-limiting step, but for the N-substituted ester and the amide acylation is the slowest step. It is shown that ITC is able to produce quality kinetic data and is particularly well suited for those enzymatic reactions that cannot be measured by absorption or fluorescence spectroscopy.

  10. Trypsin and N-aminopeptidase (APN) activities in the hepatopancreas of an intertidal euryhaline crab: Biochemical characteristics and differential modulation by histamine and salinity.

    PubMed

    Michiels, Maria Soledad; Del Valle, Juana Cristina; López Mañanes, Alejandra A

    2017-02-01

    No studies are available about biochemical characteristics and modulation (i.e. by endogenous and/or environmental cues) of trypsin (a key digestive endoprotease) in hepatopancreas of intertidal euryhaline crabs neither on the possible concomitant modulation of key ectoproteases such as aminopeptidase-N (APN) involved in final steps of protein digestion. Furthermore, nothing is still known in decapods crustaceans about the role of histamine as primary chemical messenger for modulation of main components of digestive process (i.e. proteases). We determined biochemical characteristics and investigated the effect of histamine injections; of histamine in vitro and of acclimation of individuals to low and high salinity on trypsin and aminopeptidase-N (APN) activities in the hepatopancreas of the euryhaline crab Cyrtograpsus angulatus (Dana 1851). Trypsin activity was maximal at pH7.4 and at 45°C. APN activity increased from pH6.6 to 7.6-9.0 and was maintained high at 37-45°C. Both activities exhibited Michaelis-Menten kinetics (apparent Km: trypsin=0.36mM; APN=0.07mM). The injection of 10(-4)M histamine decreased trypsin activity (about 40%) in hepatopancreas while did not affect APN activity. Similarly, in vitro 10(-4)M histamine decreased trypsin activity (about 52%) in hepatopancreas but not APN activity. Trypsin activity in the hepatopancreas was not affected by acclimation of crabs to low (10psu) or high (40psu) environmental salinity while APN activity was increased (about 200%) in 10psu. The results show the differential modulation of trypsin and APN by distinct cues and point to histamine as modulator of intracellular trypsin by direct action on the hepatopancreas.

  11. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

    PubMed

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

    2015-08-01

    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

  12. Expression and purification of a cold-adapted group III trypsin in Escherichia coli.

    PubMed

    Pálsdóttir, Helga Margrét; Gudmundsdóttir, Agústa

    2007-02-01

    The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.

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

    NASA Astrophysics Data System (ADS)

    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

  14. Expression of a cold-adapted fish trypsin in Pichia pastoris.

    PubMed

    Macouzet, Martin; Simpson, Benjamin K; Lee, Byong H

    2005-06-01

    Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.

  15. Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

    PubMed

    Hurley, Michael J; Durrenberger, Pascal F; Gentleman, Steve M; Walls, Andrew F; Dexter, David T

    2015-09-01

    Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in

  16. Thiaminase activity in native freshwater mussels

    USGS Publications Warehouse

    Blakeslee, Carrie J.; Sweet, Stephanie; Galbraith, Heather S.; Honeyfield, Dale C.

    2015-01-01

    Thiamine (vitamin B1) deficiency in the Great Lakes has been attributed to elevated levels of thiaminase I enzyme activity in invasive prey species; however, few studies have investigated thiaminase activity in native prey species. Some of the highest levels of thiaminase activity have been measured in invasive dreissenid mussels with little understanding of background levels contributed by native freshwater mussels (Bivalvia: Unionidae). In this study, thiaminase activity was measured in two freshwater mussel species, Elliptio complanata and Strophitus undulatus, from the Delaware and Susquehanna River drainage basins located in north eastern United States. Thiaminase activity was also measured in gravid and non-gravid S. undulatus. Average thiaminase activity differed significantly between species (7.2 and 42.4 μmol/g/min, for E. complanata and S. undulatus respectively) with no differences observed between drainage basins. Gravid S. undulatus had significantly lower thiaminase activity (28.0 μmol/g/min) than non-gravid mussels (42.4 μmol/g/min). Our results suggest that a suite of factors may regulate thiaminase activity in freshwater mussels and that native freshwater mussel thiaminase activity is within the range observed for invasive dreissenids. These results add to our understanding of the complexities in identifying the ecological conditions that set the stage for thiamine deficiency.

  17. Brown Kidney Bean Bowman-Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity.

    PubMed

    Chan, Yau Sang; Zhang, Yanbo; Ng, Tzi Bun

    2013-02-01

    A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. 'brown kidney bean'. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman-Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0-14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC(50) of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.

  18. Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

    PubMed

    Li, Miao; Liu, Qin; Cui, Yajuan; Li, Dong; Wang, Hexiang; Ng, Tzi Bun

    2017-01-23

    A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 μM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 μM and 2.5 μM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 μM.

  19. Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity*

    PubMed Central

    Sorensen, Anders B.; Madsen, Jesper J.; Svensson, L. Anders; Pedersen, Anette A.; Østergaard, Henrik; Overgaard, Michael T.; Olsen, Ole H.; Gandhi, Prafull S.

    2016-01-01

    The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity. PMID:26694616

  20. Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation

    PubMed Central

    Song, Feifei; Chen, Chen; Wu, Songqing; Shao, Ensi; Li, Mengnan; Guan, Xiong; Huang, Zhipeng

    2016-01-01

    Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura. PMID:27025647

  1. Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation.

    PubMed

    Song, Feifei; Chen, Chen; Wu, Songqing; Shao, Ensi; Li, Mengnan; Guan, Xiong; Huang, Zhipeng

    2016-03-30

    Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura.

  2. Introduction of {alpha}-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

    SciTech Connect

    Zablotna, Ewa; Kret, Agnieszka; Jaskiewicz, Anna; Olma, Aleksandra; Leplawy, Miroslaw T.; Rolka, Krzysztof . E-mail: krzys@chem.univ.gda.pl

    2006-02-17

    In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P{sub 1}-P{sub 1}{sup '} reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P{sub 1} by {alpha}-hydroxymethylserine (HmSer) and both enantiomers of {alpha}-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine {beta}-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine {beta}-trypsin, whereas [Val{sup 5}]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser{sup 5}]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated {epsilon}-NH{sub 2} group of the inhibitor P{sub 1} position and {beta}-carboxylate of trypsin Asp{sup 189} is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.

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

    PubMed

    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.

  4. Zymogen activation confers thermodynamic stability on a key peptide bond and protects human cationic trypsin from degradation.

    PubMed

    Szabó, András; Radisky, Evette S; Sahin-Tóth, Miklós

    2014-02-21

    Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.

  5. Engineering trypsin for inhibitor resistance.

    PubMed

    Batt, Anna R; St Germain, Commodore P; Gokey, Trevor; Guliaev, Anton B; Baird, Teaster

    2015-09-01

    The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM  = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

  6. Anti-autolysis of trypsin by modification of autolytic site Arg117.

    PubMed

    Li, X F; Nie, X; Tang, J G

    1998-09-18

    In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.

  7. Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

    PubMed Central

    Swathi, Marri; Mishra, Prashant K.; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2016-01-01

    Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus. PMID:27656149

  8. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- During a pre-launch Native American ceremony, Radmilla Cody, the 2001 Miss Navaho Nation, sings the 'Star Spangled Banner' in her native language. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

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

    PubMed

    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.

  10. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981), p... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme...

  11. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981),...

  12. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981),...

  13. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981),...

  14. Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins.

    PubMed

    Yan, Xiuwen; Liu, Huan; Yang, Xuening; Che, Qiaolin; Liu, Rui; Yang, Hailong; Liu, Xiuhong; You, Dewen; Wang, Aili; Li, Jianxu; Lai, Ren

    2012-07-01

    Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.

  15. Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives.

    PubMed Central

    Nureddin, A; Inagami, T

    1975-01-01

    1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p

  16. A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract

    PubMed Central

    2011-01-01

    Background Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. Methods The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools. Results The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein

  17. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- During a pre-launch Native American ceremony, Radmilla Cody (right) , the 2001 Miss Navaho Nation, sings the 'Star Spangled Banner' in her native language. With her is her grandmother. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  18. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. - An elder of her Navaho tribe, Dorothy Cody shares the stage with her granddaughter Radmilla Cody (not shown), the 2001 Miss Navaho Nation, who is singing the 'Star Spangled Banner' in her native language during a pre-launch Native American ceremony. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  19. Engineering the oxyanion hole of trypsin for promoting the reverse of proteolysis.

    PubMed

    Franke, Lars; Liebscher, Sandra; Bordusa, Frank

    2014-02-01

    Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate.

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

    PubMed

    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.

  1. Trypsin Inhibitor in Mung Bean Cotyledons

    PubMed Central

    Chrispeels, Maarten J.; Baumgartner, Bruno

    1978-01-01

    Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings. The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts. The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown. ImagesFig. 2Fig. 3Fig. 4Fig. 9 PMID:16660348

  2. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- The Chickasaw Dance Troupe performs an Honor Dance for John Herrington's parents during the Native American Ceremony at the Rocket Garden in the KSC Visitor Complex. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  3. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- The Chickasaw Dance Troupe performs an Honor Dance during the Native American Ceremony at the Rocket Garden in the KSC Visitor Complex. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  4. Trypsinization-dependent cell labeling with fluorescent nanoparticles

    PubMed Central

    2014-01-01

    Trypsin is often used to detach adhered cell subculture from a substrate. However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins. The present study shows that cellular uptake of fluorescent nanoparticles is remarkably increased within 24 h after trypsinization. These results highlight the trypsin-induced protein digestion, provoking leaky cell plasma membrane which leads to the strongly enhanced cellular uptake of the nanoparticles. To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization. PMID:25328505

  5. Characterization of the Interaction Between Pancreatic Trypsin and an Enteric Copolymer as a Tool for Several Biotechnological Applications.

    PubMed

    Braia, Mauricio Javier; Loureiro, Dana Belén; Tubio, Gisela; Romanini, Diana

    2015-12-01

    Protein-polyelectrolyte complexes are very interesting systems since they can be applied in many long-established and emerging areas of biotechnology. From nanotechnology to industrial processing, these complexes are used for many purposes: to build multilayer particles for biosensors; to entrap and deliver proteins for pharmaceutical applications; to isolate and immobilize proteins. The enteric copolymer poly(methacrylic acid-co-methyl methacrylate) 1:2 (MMA) has been designed for drug delivery although its chemical properties allow to use it for other applications. Understanding the interaction between trypsin and this polymer is very important in order to optimize the mechanism of formation of this complex for different biotechnological applications.The formation of the trypsin-MMA complex was studied by spectroscopy and isothermal titration calorimetry. Structural analysis of trypsin was carried out by catalytic activity assays, circular dichroism and differential scanning calorimetry. Isothermal titration calorimetry experiments showed that the insoluble complex contains 12 trypsin molecules per MMA molecule at pH 5 and they interact with high affinity to form insoluble complexes. Both electrostatic and hydrophobic forces are involved in the formation of the complex. The structure of trypsin is not affected by the presence of MMA, although it interacts with some domains of trypsin affecting its thermal denaturation as seen in the differential scanning calorimetry experiments. Its catalytic activity is not altered. Dynamic light scattering demonstrated the presence of a soluble trypsin-copolymer complex at pH 5 and 8. Turbidimetric assays show that the insoluble complex can be dissolved by low ionic strength and/or pH in order to obtain free native trypsin.

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

    PubMed

    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.

  7. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Seminole Native American Veterans serve as color guard during a pre-launch Native American ceremony at the Rocket Garden in the KSC Visitor Complex. David Nunez, U.S. Navy, carries the State of Florida Flag; David Stephen Bowers, U.S. Army, carries the Flag of the United States of America; Charles Billie Hiers, U.S. Marine Corps., carries the Seminole Tribe of Florida Flag. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  8. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Chickasaw Indian princesses seen here contributed to a pre-launch Native American ceremony at the Rocket Garden in the KSC Visitor Complex by leading a prayer. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  9. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Singer Buffy Sainte-Marie sings during a pre-launch Native American ceremony in the Rocket Garden of the KSC Visitor Complex. She herself is a Cree. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  10. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Chickasaw Indian princesses pose with folk singer Buffy Saint- Marie (center) during a Native American ceremony held in the Rocket Garden in the KSC Visitor Complex. Several days of activities were held at KSC and in Orlando commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  11. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. - Chickasaw Indian princesses 'sign' the Lord's Prayer during a Native American Ceremony at the Rocket Garden in the KSC Visitor Complex. The princesses are Crystal Underwood, Julie Underwood and Tamela Alexander. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  12. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Chickasaw Tribal Elder Lee Frazier leads the dedication to the astronauts of STS-113 during the Native American Ceremony at the Rocket Garden in the KSC Visitor Complex. The ceremony was part of several days' activities commemorating John B. Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission. Herrington is a Mission Specialist on STS-113.

  13. Interesting biological activities from plants traditionally used by Native Australians.

    PubMed

    Pennacchio, Marcello; Kemp, Annabeth S; Taylor, Rory P; Wickens, Kristen M; Kienow, Lucie

    2005-01-15

    Four plants routinely used for medicinal purposes by Native Australians were screened for various biological activities. Methanol extracts of Eremophila maculata, Acacia auriculoformis and Acacia bivenosa exhibited antibiotic effects, while Eremophila alternifolia yielded an extract that induced significant changes to the heart activity of spontaneously hypertensive rats. We report on these biological activities.

  14. Chitosan nanoparticles conjugate with trypsin and trypsin inhibitor.

    PubMed

    Chanphai, P; Tajmir-Riahi, H A

    2016-06-25

    Chitosan-protein conjugates are widely used in therapeutic drug delivery. We report the bindings of chitosan nanoparticles with trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis and multiple spectroscopic methods. Thermodynamic parameters ΔS, ΔH and ΔG showed chitosan-protein bindings occur mainly via H-bonding and van der Waals contacts with trypsin inhibitor forming more stable conjugate than trypsin. As chitosan size increased more stable polymer-protein conjugate was formed. Chitosan complexation induces more perturbations of trypsin inhibitor structure than trypsin with reduction of protein alpha-helix and major increase of random structure. The negative value of ΔG indicates spontaneous protein-chitosan complexation at room temperature. Chitosan nanoparticles can be used to transport trypsin and trypsin inhibitor.

  15. Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency.

    PubMed

    Perera, Erick; Rodríguez-Casariego, Javier; Rodríguez-Viera, Leandro; Calero, Jorge; Perdomo-Morales, Rolando; Mancera, Juan M

    2012-04-01

    It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.

  16. Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

    PubMed Central

    Terras, FRG.; Schoofs, HME.; Thevissen, K.; Osborn, R. W.; Vanderleyden, J.; Cammue, BPA.; Broekaert, W. F.

    1993-01-01

    Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes. PMID:12232024

  17. Polymer masked-unmasked protein therapy. 1. Bioresponsive dextrin-trypsin and -melanocyte stimulating hormone conjugates designed for alpha-amylase activation.

    PubMed

    Duncan, Ruth; Gilbert, Helena R P; Carbajo, Rodrigo J; Vicent, María J

    2008-04-01

    Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The

  18. Protective effect of Carbopol on enzymatic degradation of a peptide-like substrate. I: Effect of various concentrations and grades of Carbopol and other reaction variables on trypsin activity.

    PubMed

    Vaidya, A P; Wigent, R J; Moore, J C; Schwartz, J B

    2007-01-01

    The purpose of this study was to determine and compare the effect of various concentrations and grades of Carbopol on trypsin-induced degradation of a prototype substrate, N(alpha)-benzoyl-L-arginine ethyl ester hydrochloride (BAEE). Effect of other reaction variables, such as viscosity and ionic strength of the medium on the trypsin activity, was also analyzed simultaneously. Four concentrations and three commercially available grades of Carbopol were used. The effect of Carbopol was expressed in terms of change in the velocity of degradation reaction. A modified trypsin assay was developed and used for analysis. Up to a concentration of 0.35% w/v, Carbopol 934P showed a concentration-dependent increase in its ability to reduce the rate of enzymatic hydrolysis of BAEE. Similar inhibitory effect was observed with all three grades of Carbopol. The activity of trypsin was unaffected by other reaction variables, suggesting that interaction between the protein and the polymer could be the mechanism responsible for reduced trypsin activity. This study suggests that Carbopol can be a useful excipient for oral delivery of bioactive proteins and peptides, due to its ability to reduce the enzyme-induced degradation of these agents.

  19. Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

    PubMed

    Castellanos-Moguel, Judith; González-Barajas, Margarita; Mier, Teresa; Reyes-Montes, María Del Rocío; Aranda, Eduardo; Toriello, Conchita

    2007-03-01

    To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

  20. A rational design for improving the trypsin resistance of aflatoxin-detoxifizyme (ADTZ) based on molecular structure evaluation.

    PubMed

    Qiu, Yuxin; Wu, Xiyang; Xie, Chunfang; Hu, Yadong; Liu, Daling; Ma, Yi; Yao, Dongsheng

    2016-05-01

    The resistance of feed enzymes against proteases is crucial in livestock farming. In this study, the trypsin resistance of aflatoxin-detoxifizyme (ADTZ) is improved. ADTZ possesses 72 lys/arg residue sites, 45 of which are scattered on the outermost layers of the molecule (RSA≧25%). These 45 lys/arg sites could be target sites for trypsin hydrolysis. By considering shape-matching (including physical and secondary bond interactions) and the "induced fit-effect", we hypothesized that some of these lys/arg sites are vulnerable to trypsin. A protein-protein docking simulation method was used to avoid the massive computational requirements and to address the intricacy of selecting candidate sites, as candidate site selection is affected by space displacement. Optimal mutants (K244Q/K213C/K270T and R356E/K357T/R623C) were predicted by computational design with protein folding energy analysis and molecular dynamics simulations. A trypsin digestion assay was performed, and the mutants displayed much higher stability against trypsin hydrolysis compared to the native enzyme. Moreover, temperature- and pH-activity profiles revealed that the designed mutations did not affect the catalytic activity of the enzyme.

  1. Combinatorial variation in coding and promoter sequences of genes at the Tri locus in Pisum sativum accounts for variation in trypsin inhibitor activity in seeds.

    PubMed

    Page, D; Aubert, G; Duc, G; Welham, T; Domoney, C

    2002-05-01

    Cultivars of Pisum sativum that differ with respect to the quantitative expression of trypsin/chymotrypsin inhibitor proteins in seeds have been examined in terms of the structure of the corresponding genes. The patterns of divergence in the promoter and coding sequences are described, and the divergence among these exploited for the development of facile DNA-based assays to distinguish genotypes. Quantitative effects on gene expression may be attributed to the overall gene complement and to particular promoter/coding sequence combinations, as well as to the existence of distinct active-site variants that ultimately influence protein activity. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00438-002-0667-4.

  2. A laundry detergent-stable alkaline trypsin from striped seabream (Lithognathus mormyrus) viscera: purification and characterization.

    PubMed

    El Hadj Ali, Nedra; Hmidet, Noomen; Bougatef, Ali; Nasri, Rim; Nasri, Moncef

    2009-11-25

    An alkaline trypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified and characterized. The enzyme was purified to homogeneity by precipitation with ammonium sulfate, Sephadex G-100 gel filtration and CM-Sephadex cation-exchange chromatography, with a 24.9-fold increase in specific activity and 13% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The purified trypsin appeared as a single band on native PAGE. Interestingly, the enzyme was highly active over a wide range of pH from 8.0 to 11.0, with an optimum at pH 10.0 using Nalpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 8.0, 11.0, and 12.0 were 73%, 67% and 50.4%, respectively. The enzyme was extremely stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 degrees C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI). In addition, the enzyme showed excellent stability toward various surfactants and bleache agents and compatibility with some commercial solid and liquid detergents. The trypsin kinetic constants, Km and kcat of the enzyme for BAPNA, were 0.29 mM and 1.36 s(-1), respectively, while the catalytic efficiency kcat/Km was 4.68 s(-1) mM(-1).

  3. Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes.

    PubMed

    Stefansson, Bjarki; Sandholt, Gunnar B; Gudmundsdottir, Ágústa

    2017-01-01

    Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.

  4. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

    PubMed Central

    2010-01-01

    Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea). M. excelsa (Pohutukawa) was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity. PMID:20537175

  5. Inactivation of pectin methylesterase by immobilized trypsins from cunner fish and bovine pancreas.

    PubMed

    Li, Dan; Matos, Madyu; Simpson, Benjamin K

    2013-01-01

    Immobilized cunner fish trypsin was used to inactivate pectin methylesterase (PME). The effects of different reaction conditions (e.g., incubation time, PME concentration, and temperature) on PME inactivation and kinetics of inactivation were investigated. Temperature, incubation time, and PME concentration significantly affected the extent of PME inactivation. Generally, higher temperature, longer incubation time, and low PME concentration caused more PME inactivation. The immobilized fish trypsin had higher capacity to inactivate PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was about 20% higher than that of its bovine counterpart. However, PME inactivated by both trypsins regained partial activity during storage at 4°C, with immobilized fish trypsin-treated PME regaining more of its original activity than the immobilized bovine trypsin-treated PME. Heat-denatured PME was hydrolyzed more extensively by immobilized fish trypsin than by its bovine counterpart. The rate constants increased, whereas the D-values decreased with temperature for both immobilized fish and bovine trypsins. The inactivation rate constants of immobilized fish trypsin at all the temperatures investigated (i.e., 15-35°C) were higher than those of immobilized bovine trypsin. Furthermore, the activation energy (Ea ) of PME inactivation by immobilized fish trypsin was lower than that of immobilized bovine trypsin.

  6. Molecular sequencing and modeling of Neobellieria bullata trypsin. Evidence for translational control by Neobellieria trypsin-modulating oostatic factor.

    PubMed

    Borovsky, D; Janssen, I; Vanden Broeck, J; Huybrechts, R; Verhaert, P; De Bondt, H L; Bylemans, D; De Loof, A

    1996-04-01

    Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.

  7. Binding characteristics of psoralen with trypsin: Insights from spectroscopic and molecular modeling studies.

    PubMed

    Liu, Yingying; Zhang, Guowen; Liao, Yijing; Wang, Yaping

    2015-01-01

    Psoralen (PSO) is a naturally occurring furanocoumarin with a variety of pharmacological activities, however very limited information on the interaction of PSO with trypsin is available. In this study, the binding characteristics between PSO and trypsin at physiological pH were investigated using a combination of fluorescence, UV-vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic, chemometric and molecular modeling approaches. It was found that the fluorescence quenching of trypsin by PSO was a static quenching procedure, ascribing the formation of a PSO-trypsin complex. The binding of PSO to trypsin was driven mainly by hydrophobic forces as the positive enthalpy change and entropy change values. The molecular docking showed that PSO inserted into the active site pocket of trypsin to interact with the catalytic residues His57, Asp102 and Ser195 and may cause a decrease in trypsin activity. The results of CD and FT-IR spectra along with the temperature-induced denaturation studies indicated that the addition of PSO to trypsin led to the changes in the secondary structure of the enzyme. The concentration profiles and spectra of the three components (PSO, trypsin, and PSO-trypsin complex) obtained by multivariate curve resolution-alternating least squares analysis exhibited the kinetic processes of PSO-trypsin interaction. This study will be helpful to understand the mechanism of PSO that affects the conformation and activity of trypsin in biological processes.

  8. Micropollutant degradation via extracted native enzymes from activated sludge.

    PubMed

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  9. Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

    PubMed

    Esparza, I; Brock, J H

    1978-01-01

    1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.

  10. A comparison of kaolin-activated versus nonkaolin-activated thromboelastography in native and citrated blood.

    PubMed

    Thalheimer, Ulrich; Triantos, Christos K; Samonakis, Dimitrios N; Zambruni, Andrea; Senzolo, Marco; Leandro, Gioacchino; Patch, David; Burroughs, Andrew K

    2008-09-01

    Thromboelastography can be performed with native or citrated blood (a surrogate to native blood in healthy controls, surgical and cirrhotic patients). Activators such as kaolin are increasingly used to reduce the time to trace generation. To compare kaolin-activated thromboelastography with nonkaolin-activated thromboelastography of native and citrated blood in patients with liver disease, patients undergoing treatment with warfarin or low-molecular weight heparin and healthy volunteers. We studied thromboelastography parameters in 21 healthy volunteers (group 1) and 50 patients, including 20 patients with liver cirrhosis with a nonbiliary aetiology (group 2), 10 patients with primary biliary cirrhosis or primary sclerosing cholangitis (group 3), 10 patients on warfarin treatment (group 4) and 10 patients with enoxaparin prophylaxis (group 5). Thromboelastography was performed using four methods: native blood (kaolin-activated and nonkaolin-activated) and citrated blood (kaolin-activated and nonkaolin-activated). For all thromboelastography parameters, correlation was poor (Spearman correlation coefficient < 0.70) between nonkaolin-activated and kaolin-activated thromboelastography, for both citrated and native blood. In healthy volunteers, in patients with liver disease and in those receiving anticoagulant treatment, there was a poor correlation between nonkaolin-activated and kaolin-activated thromboelastography. Kaolin-activated thromboelastography needs further validation before routine clinical use in these settings, and the specific methodology must be considered in comparing published studies.

  11. Protease inhibitors in various flours and breads: Effect of fermentation, baking and in vitro digestion on trypsin and chymotrypsin inhibitory activities.

    PubMed

    Kostekli, Mine; Karakaya, Sibel

    2017-06-01

    In this study trypsin (TIA) and chymotrypsin inhibitory (CIA) activities were determined in the extracts of wheat, rye mix, mixed cereals and, whole wheat flours and, breads made with these flours. In addition, effects of fermentation, baking and in vitro digestion on TIA and CIA were studied. Whole wheat flour, dough, and bread did not show any TIA. Other flours displayed TIA. Contrary to, all flours, doughs, and breads exhibited CIA. Although TIA was not detected in the protein extracts obtained from wheat and rye mix breads, protein extract of rye mix flour exhibited TIA. Following in vitro digestion process, TIA of wheat bread was found as 5.91units/mL gastric dialysate and 9.17units/mL intestine dialysate. CIA was determined in dialysates obtained from wheat (2.12CI/mL and 3.78CI/mL for gastric and intestinal dialysate respectively) and rye breads (4.16CI/mL and 2.46CI/mL for gastric and intestinal dialysate respectively).

  12. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... activity of trypsin (a pancreatic enzyme important in digestion for the breakdown of proteins) in blood...

  13. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... activity of trypsin (a pancreatic enzyme important in digestion for the breakdown of proteins) in blood...

  14. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... activity of trypsin (a pancreatic enzyme important in digestion for the breakdown of proteins) in blood...

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

    NASA Astrophysics Data System (ADS)

    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.

  16. In vivo bioinsecticidal activity toward Ceratitis capitata (fruit fly) and Callosobruchus maculatus (cowpea weevil) and in vitro bioinsecticidal activity toward different orders of insect pests of a trypsin inhibitor purified from tamarind tree (Tamarindus indica) seeds.

    PubMed

    Araújo, Carina L; Bezerra, Ingrid W L; Oliveira, Adeliana S; Moura, Fabiano T; Macedo, Leonardo L P; Gomes, Carlos E M; Barbosa, Aulus E A D; Macedo, Francisco P; Souza, Tánia M S; Franco, Octavio L; Bloch-J, Carlos; Sales, Mauricio P

    2005-06-01

    A proteinaceous inhibitor with high activity against trypsin-like serine proteinases was purified from seeds of the tamarind tree (Tamarindus indica) by gel filtration on Shephacryl S-200 followed by a reverse-phase HPLC Vidac C18 TP. The inhibitor, called the tamarind trypsin inhibitor (TTI), showed a Mr of 21.42 kDa by mass spectrometry analysis. TTI was a noncompetitive inhibitor with a Ki value of 1.7 x 10(-9) M. In vitro bioinsecticidal activity against insect digestive enzymes from different orders showed that TTI had remarkable activity against enzymes from coleopteran, Anthonomus grandis (29.6%), Zabrotes subfasciatus (51.6%), Callosobruchus maculatus (86.7%), Rhyzopertha dominica(88.2%), and lepidopteron, Plodia interpuncptella (26.7%), Alabama argillacea (53.8%), and Spodoptera frugiperda (75.5%). Also, digestive enzymes from Diptera, Ceratitis capitata (fruit fly), were inhibited (52.9%). In vivo bioinsecticidal assays toward C. capitata and C. maculatus larvae were developed. The concentration of TTI (w/w) in the artificial seed necessary to cause 50% mortality (LD50) of larvae was 3.6%, and that to reduce mass larvae by 50.0% (ED50) was 3.2%. Furthermore, the mass C. capitata larvae were affected at 53.2% and produced approximately 34% mortality at a level of 4.0% (w/w) of TTI incorporated in artificial diets.

  17. Possible conformational change within the desolvated and cationized sBBI/trypsin non-covalent complex during the collision-induced dissociation process.

    PubMed

    Darii, Ekaterina; Saravanamuthu, Gunalini; Afonso, Carlos; Alves, Sandra; Gut, Ivo; Tabet, Jean-Claude

    2011-06-30

    Electrospray ionization mass spectrometry (ESI-MS) has become an analytical technique widely used for the investigation of non-covalent protein-protein and protein-ligand complexes due to the soft desolvation conditions that preserve the stoichiometry of the interacting partners. Dissociation studies of solvated or desolvated complexes (in the source and in the collision cell, respectively) allow access to information on protein conformation and localization of the metal ions involved in protein structure stabilization and biological activity. The complex of bovine trypsin and small soybean Bowman-Birk inhibitor (sBBI) was studied by ESI-MS to determine changes occurring within the complex during its transfer from droplets to the gas phase independently of the ion polarity. Under collision-induced dissociation (CID) conditions, unexpected binding of the Ca(2+) ion (cofactor of native trypsin) to the inhibitor molecule was observed within the desolvated sBBI/trypsin/Ca(2+) complex (with a 1:1:1 stoichiometry). This formal gas-phase migration of the calcium ion from trypsin to the inhibitor may be related to conformational rearrangements in the solvent-free and likely collapsed complex. However, under conditions leading to the increase in complex charge state, the appearance of the cationized trypsin molecule was detected during complex dissociation, thus reflecting different pathways of the evolution of complex conformation.

  18. Characterization and expression of trypsinogen and trypsin in medaka testis.

    PubMed

    Rajapakse, Sanath; Ogiwara, Katsueki; Takahashi, Takayki

    2014-12-01

    Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.

  19. Purification and characterization of a stable Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds.

    PubMed

    Oddepally, Rajender; Sriram, Gopi; Guruprasad, Lalitha

    2013-12-01

    Kunitz trypsin inhibitor was purified from the seeds of Trigonella foenum-graecum (TfgKTI) belonging to fabaceae family by ammonium sulphate precipitation, cation exchange, gel filtration and hydrophobic chromatography. Purity of the protein was analyzed by RP-HPLC and native-PAGE. SDS-PAGE analysis under reducing and non-reducing conditions showed that protein consists of a single polypeptide chain with molecular mass of approximately 20 kDa. Mass spectroscopy analysis revealed that the intact mass of purified inhibitor is 19,842.154 Da. One dimensional SDS gel was tryptically digested, resulting peptides were subjected to MALDI-TOF-MS analysis, and peptide mass fingerprinting (PMF) analysis of TfgKTI shows sequence similarity with Kunitz trypsin inhibitor in database search. Two dimensional electrophoresis identified presence of four isoinhibitors (pI values of 5.1, 5.4, 5.7 and 6.1). Kinetic studies showed that the protein is a competitive inhibitor and has high binding affinity with trypsin (Ki 3.01×10(-9)M) and chymotrypsin (Ki 0.52×10(-9)M). The TfgKTI retained the inhibitory activity over a broad range of pH (pH 3-10), temperature (37-100°C) and salt concentration (up to 3.5%). Far-UV circular dichroism measurements revealed that TfgKTI is predominantly composed of β-sheets (39%) and unordered structures (48%) with slight helical content (13%). TfgKTI retained over 90% trypsin inhibition upon storage at 4°C for over a period of six months.

  20. Trypsin purification using magnetic particles of azocasein-iron composite.

    PubMed

    Alves, Maria Helena Menezes Estevam; Nascimento, Gabriela Ayres; Cabrera, Mariana Paola; Silvério, Sara Isabel da Cruz; Nobre, Clarisse; Teixeira, José António; de Carvalho, Luiz Bezerra

    2017-07-01

    This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

  1. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Joyce and James Herrington, parents of John Herrington, accept a gift during a pre-launch Native American ceremony. They are the parents of John Herrington, mission specialist on mission STS-113. Herrington is the first Native American to be going into space.

  2. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. - Chickasaw Dance troupe member Tim Harjo (second from left) leads Joyce and James Herrington in a dance honoring their son, STS-113 Mission Specialist John Herrington. The dance was part of a Native American ceremony at the Rocket Garden in the KSC Visitor Complex commemorating Herrington as the first tribally enrolled Native American astronaut to fly on a Shuttle mission.

  3. An improved trypsin digestion method minimizes digestion-induced modifications on proteins.

    PubMed

    Ren, Da; Pipes, Gary D; Liu, Dingjiang; Shih, Liang-Yu; Nichols, Andrew C; Treuheit, Michael J; Brems, David N; Bondarenko, Pavel V

    2009-09-01

    Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.

  4. On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+)-activated K+ channels. A model system for analysis of peptide- induced subconductance states

    PubMed Central

    1991-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation. PMID:1714938

  5. Antioxidant activity in Australian native sarsaparilla (Smilax glyciphylla).

    PubMed

    Cox, Sean D; Jayasinghe, K Chamila; Markham, Julie L

    2005-10-03

    A hot water extract of the Australian native sarsaparilla Smilax glyciphylla Sm. (Smilaceae) inhibited peroxidation of phosphatidylcholine liposomes initiated by Fe(2+)/ascorbate (IC50, 10 microg/mL) and AAPH (IC50, 33 microg/mL) in vitro. It also inhibited deoxyribose degradation and quenched chemically generated superoxide anion (IC50, 50 microg/mL). Reactivity towards ABTS (2,2'-azinobis(3-ethylbenzothiazoline 6-sulphonate) radical cation was equivalent to 48.4 mM TROLOX, the water soluble alpha-tocopherol analogue. Smilax glyciphylla is a rich source of the dihydrochalcone glycyphyllin. Given the reported level of activity it is unlikely that glycyphyllin would provide direct antioxidant protection in tissues affected by oxidative stress. However, consuming Smilax glyciphylla as a tea may be sufficient to reduce oxidative damage in the gastrointestinal tract. It is also possible that glycyphyllin is metabolised and adsorbed as phloretin, a compound with known anticancer properties. These findings indicate that further studies of the chemopreventative properties of Smilax glyciphylla is warranted.

  6. The NESA Activities Handbook for Native and Multicultural Classrooms. Volume Two.

    ERIC Educational Resources Information Center

    Sawyer, Don, Comp.; Napoleon, Art, Comp.

    This book, the second of three volumes, contains educational, culture-sensitive activities tested and designed for use in native and multicultural classrooms. The activities, developed Native Education Services Associates, stress the importance of culture in students' lives, and teaches them basic personal and community-related skills so they may…

  7. Interaction between 8-methoxypsoralen and trypsin: Monitoring by spectroscopic, chemometrics and molecular docking approaches

    NASA Astrophysics Data System (ADS)

    Liu, Yingying; Zhang, Guowen; Zeng, Ni; Hu, Song

    2017-02-01

    8-Methoxypsoralen (8-MOP) is a naturally occurring furanocoumarin with various biological activities. However, there is little information on the binding mechanism of 8-MOP with trypsin. Here, the interaction between 8-MOP and trypsin in vitro was determined by multi-spectroscopic methods combined with the multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics approach. An expanded UV-vis spectral data matrix was analysed by MCR-ALS, the concentration profiles and pure spectra for the three reaction species (trypsin, 8-MOP and 8-MOP-trypsin) were obtained to monitor the interaction between 8-MOP and trypsin. The fluorescence data suggested that a static type of quenching mechanism occurred in the binding of 8-MOP to trypsin. Hydrophobic interaction dominated the formation of the 8-MOP-trypsin complex on account of the positive enthalpy and entropy changes, and trypsin had one high affinity binding site for 8-MOP with a binding constant of 3.81 × 104 L mol- 1 at 298 K. Analysis of three dimensional fluorescence, UV-vis absorption and circular dichroism spectra indicated that the addition of 8-MOP induced the rearrangement of the polypeptides carbonyl hydrogen-bonding network and the conformational changes in trypsin. The molecular docking predicted that 8-MOP interacted with the catalytic residues His57, Asp102 and Ser195 in trypsin. The binding patterns and trypsin conformational changes may result in the inhibition of trypsin activity. This study has provided insights into the binding mechanism of 8-MOP with trypsin.

  8. Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

    PubMed

    Woodard, Susan L; Mayor, Jocelyne M; Bailey, Michele R; Barker, Donna K; Love, Robert T; Lane, Jeffrey R; Delaney, Donna E; McComas-Wagner, Janet M; Mallubhotla, Hanuman D; Hood, Elizabeth E; Dangott, Lawrence J; Tichy, Shane E; Howard, John A

    2003-10-01

    Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

  9. Porphyrin Induced Laser Deactivation of Trypsinogen-Trypsin Conversion

    NASA Astrophysics Data System (ADS)

    Perido, Joanna; Brancaleon, Lorenzo

    2015-03-01

    Pancreatitis is caused by the inflammation of the pancreas, where the digestive enzyme trypsin is activated from the precursor enzyme trypsinogen while still in the pancreas. The presence of trypsin in the pancreas causes auto-activation of trypsinogen, resulting in greater inflammation and auto-digestion of the pancreas. In severe cases, this cascade effect can lead to organ failure, diabetes, and pancreatic cancer. Our hypothesis is that if trypsinogen is prevented from auto-activating into trypsin, then this cascade can be stopped. We propose to do this by inducing conformational changes in trypsinogen when bound to a photoactive porphyrin dye. Porphyrins are comprised of four linked heterocyclic groups forming a flat ring, and bind well with proteins such as trypsinogen. In this study we used spectroscopic techniques to probe the binding of meso-tetrakis (4-sulfonatephenyl) porphyrin (TSPP) to trypsinogen in vitro, as a preliminary step to then prompt and characterize conformational changes of trypsinogen through irradiation. If conformational changes are detected the trypsinogen will be tested for trypsin inactivation. This investigation may provide promising initial results to the possible use of porphyrins as an inhibitor of the self-activation of trypsinogen into trypsin, and a potential inhibitor of pancreatitis. MARC*U-STAR.

  10. Chitosan Nanoencapsulated Exogenous Trypsin Biomimics Zymogen-Like Enzyme in Fish Gastrointestinal Tract

    PubMed Central

    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

  11. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. - Mr. and Mrs. Sean O'Keefe (center) pose with officials of the Chickasaw Nation. Second from left is Lt. Gov. Jefferson Keel with his wife, Carol (far left). Second from right is Gov. Bill Anoatubby with his wife, Janice (far right). STS-113 Mission Specialist John Herrington is a tribally enrolled Chickasaw and the world's first Native American astronaut. Kennedy Space Center hosted more than 350 Native Americans in STS-113 prelaunch events surrounding the historic mission assignment of Herrington.

  12. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- During an administrator's briefing at the IMAX 2 theatre, Lt. Gov. Jefferson Keel of the Chickasaw Nation (far left) presents a blanket with the seal of the Chickasaw Nation to NASA Administrator Sean O'Keefe (second from right). Next to O'Keefe is Chickasaw Gov. Bill Anoatubby. Next to Gov. Keel is Mrs. Laura O'Keefe. STS-113 Mission Specialist John Herrington is a tribally enrolled Chickasaw and the world's first Native American astronaut. Kennedy Space Center hosted more than 350 Native Americans in STS-113 prelaunch events surrounding the historic mission assignment of Herrington.

  13. Activities commemorating John B. Herrington as first Native American astronaut

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- Chickasaw Nation Cultural Resources Director Haskell Alexander (left) presents a gift to Joyce and James Herrington, parents of John Herrington, mission specialist on mission STS-113. Herrington is the first Native American to be going into space.

  14. Internet Activities and Developmental Predictors: Gender Differences among Digital Natives

    ERIC Educational Resources Information Center

    Johnson, Genevieve Marie

    2011-01-01

    Widespread adoption of the Internet during the past two decades has produced the first generation of digital natives. Ninety-five children (M[subscript age] = 10.4 years) completed a questionnaire that measured three clusters of variables: 1) Internet use at home and school, 2) peer, school, and home self-esteem, 3) and cognitive abilities…

  15. Structural Basis for Accelerated Cleavage of Bovine Pancreatic Trypsin Inhibitor (BPTI) by Human Mesotrypsin

    SciTech Connect

    Salameh,M.; Soares, A.; Hockla, A.; Radisky, E.

    2008-01-01

    Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.

  16. Intracellular co-localization of trypsin-2 and matrix metalloprotease-9: possible proteolytic cascade of trypsin-2, MMP-9 and enterokinase in carcinoma.

    PubMed

    Vilen, Suvi-Tuuli; Nyberg, Pia; Hukkanen, Mika; Sutinen, Meeri; Ylipalosaari, Merja; Bjartell, Anders; Paju, Annukka; Haaparanta, Virpi; Stenman, Ulf-Håkan; Sorsa, Timo; Salo, Tuula

    2008-02-15

    Tumor-associated trypsin-2 and matrix metalloprotease-9 (MMP-9) are associated with cancer, particularly with invasive squamous cell carcinomas. They require activation for catalytical competence via proteolytic cascades. One cascade is formed by enterokinase, trypsin-2 and MMP-9; enterokinase activates trypsinogen-2 to trypsin-2, which is an efficient proMMP-9 activator. We describe here that oral squamous cell carcinomas express all members of this cascade: MMP-9, trypsin-2 and enterokinase. The expression of enterokinase in a carcinoma cell line not derived from the duodenum was shown here for the first time. Enterokinase directly cleaved proMMP-9 at the Lys65-Ser66 site, but failed to activate it in vitro. We demonstrated by confocal microscopy that MMP-9 and trypsin-2 co-localized in intracellular vesicles of the carcinoma cells. This co-localization of trypsin-2 and MMP-9 resulted in intracellular proMMP-9 processing that represented fully or partially activated MMP-9. However, although both proteases were present also in various bone tumor tissues, MMP-9 and trypsin-2 never co-localized at the cellular level in these tissues. This suggests that the intracellular vesicular co-localization, storage and possible activation of these proteases may be a unique feature for aggressive epithelial tumors, such as squamous cell carcinomas, but not for tumors of mesenchymal origin.

  17. Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification.

    PubMed

    Díaz-Mendoza, M; Ortego, F; García de Lacoba, M; Magaña, C; de la Poza, M; Farinós, G P; Castañera, P; Hernández-Crespo, P

    2005-09-01

    The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the

  18. Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules.

    PubMed

    Sznajder, Anna; Jendrossek, Dieter

    2011-03-01

    A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

  19. 78 FR 32243 - Agency Information Collection Activities; Comment Request; Native American Career and Technical...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-29

    ... Career and Technical Education Program (NACTEP) Performance Reports. OMB Control Number: 1830-0573. Type... Agency Information Collection Activities; Comment Request; Native American Career and Technical Education Program (NACTEP) Performance Reports AGENCY: Office of Vocational and Adult Education (OVAE),...

  20. Kunitz-type trypsin inhibitor with high stability from Spinacia oleracea L. seeds.

    PubMed

    Kang, Zhuang; Jiang, Jia-hong; Wang, Dong; Liu, Ke; Du, Lin-fang

    2009-01-01

    The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K(i) = 2.3x10(-6) M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.

  1. The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes.

    PubMed Central

    Larsson, L J; Lindahl, P; Hallén-Sandgren, C; Björk, I

    1987-01-01

    The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an

  2. Mechanism of gold nanoparticles-induced trypsin inhibition: a multi-technique approach.

    PubMed

    Zhang, Hongmei; Cao, Jian; Wu, Shengde; Wang, Yanqing

    2014-08-01

    The binding interactions of gold nanoparticles with trypsin were investigated using multi-spectra methods and molecular modeling. The experiment data showed that trypsin modified the surface of gold nanoparticles. The fluorescence intensity of trypsin was quenched by gold nanoparticles that strongly associated with protein and induced the inhibition of enzyme activity. The electrostatic and hydrophobic interactions were the primary contributors to the binding forces between trypsin and gold nanoparticles. The covalent interactions might be also involved in the binding process. The modeling calculated results indicated that the binding site was near to the primary substrate-binding pocket and the active site of the enzyme substrate. This work elucidated the interaction mechanism of trypsin with gold nanoparticles from the theoretical and experimental angle.

  3. The relevance of cultural activities in ethnic identity among California Native American youth.

    PubMed

    Schweigman, Kurt; Soto, Claradina; Wright, Serena; Unger, Jennifer

    2011-01-01

    This study analyzed data from a large statewide sample of Native American adolescents throughout California to determine whether participation in cultural practices was associated with stronger ethnic identity. The Multigroup Ethnic Identity Measure (MEIM) scale was used to measure the ethnic identity of 945 Native American adolescents (416 male, 529 female) aged 13 - 19 across California. Respondents who participated in cultural activities including pow-wows, sweat lodge, drum group and roundhouse dance reported significantly higher Native American ethnic identity than their counterparts who did not take part in cultural activities. The association between cultural activities and ethnic identity was only significant among urban youth and not among reservation youth. Higher grades in school were associated with ethnic identity among females but not among males. Findings from this study show a strong association between cultural activities and traditional practices with tribal enculturation among Native American youth in California. Cultural-based practices to enhance Native identity could be useful to improve mental and behavioral health among Native American youth.

  4. Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

    NASA Astrophysics Data System (ADS)

    Maciel, J. C.; D. Mercês, A. A.; Cabrera, M.; Shigeyosi, W. T.; de Souza, S. D.; Olzon-Dionysio, M.; Fabris, J. D.; Cardoso, C. A.; Neri, D. F. M.; C. Silva, M. P.; Carvalho, L. B.

    2016-12-01

    It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe 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 57Fe 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 ( γFe2O3). 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 ∘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.

  5. Carbonic anhydrase activity in the red blood cells of sea level and high altitude natives.

    PubMed

    Gamboa, J; Caceda, R; Gamboa, A; Monge-C, C

    2000-01-01

    Red blood cell carbonic anhydrase (CA) activity has not been studied in high altitude natives. Because CA is an intraerythocytic enzyme and high altitude natives are polycythemic, it is important to know if the activity of CA per red cell volume is different from that of their sea level counterparts. Blood was collected from healthy subjects living in Lima (150m) and from twelve subjects from Cerro de Pasco (4330m), and hematocrit and carbonic anhydrase activity were measured. As expected, the high altitude natives had significantly higher hematocrits than the sea level controls (p = 0.0002). No difference in the CA activity per milliliter of red cells was found between the two populations. There was no correlation between the hematocrit and CA activity.

  6. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants.

    PubMed

    Wu, Xi; Singh, Atul K; Wu, Xiaoyu; Lyu, Yuan; Bhunia, Arun K; Narsimhan, Ganesan

    2016-07-01

    Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes.

  7. Trypsin inhibitors for the treatment of pancreatitis.

    PubMed

    Brandl, Trixi; Simic, Oliver; Skaanderup, Philip R; Namoto, Kenji; Berst, Frederic; Ehrhardt, Claus; Schiering, Nikolaus; Mueller, Irene; Woelcke, Julian

    2016-09-01

    Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

  8. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ....1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation obtained from purified extracts of porcine or bovine pancreas. It is a white to tan amorphous powder. Its characterizing enzyme... and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981), p....

  9. The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

    PubMed

    Di Ciero, L; Oliva, M L; Torquato, R; Köhler, P; Weder, J K; Camillo Novello, J; Sampaio, C A; Oliveira, B; Marangoni, S

    1998-11-01

    Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

  10. Probing the binding of procyanidin B3 to trypsin and pepsin: A multi-technique approach.

    PubMed

    Li, Xiangrong; Geng, Mingjiang

    2016-04-01

    Proanthocyanidins are a mixture of monomers, oligomers, and polymers of flavan-3-ols that are widely distributed in the plant kingdom. One of the most widely studied proanthocyanidins is procyanidin B3. In this study, the binding of procyanidin B3 to trypsin and pepsin was investigated using spectrofluorimetry, equilibrium microdialysis, circular dichroism (CD) spectroscopy and molecular modeling. Fluorescence experiments indicate procyanidin B3 quenches the fluorescence of trypsin/pepsin through a static process. Thermodynamic analysis reveals that procyanidin B3 binds to trypsin/pepsin is synergistically driven by enthalpy and entropy, and the major driving forces are hydrophobic, hydrogen bonding, and electrostatic interactions. Procyanidin B3 binds trypsin in a more firmly way than pepsin, and one molecule of procyanidin B3 combines with one molecule of trypsin/pepsin. The binding parameters obtained from equilibrium microdialysis are consistent with the results obtained from fluorescence spectroscopy. Synchronous fluorescence spectroscopy and CD spectroscopy show that procyanidin B3 may induce microenvironmental and conformational changes of trypsin and pepsin. Molecular modeling displays the specific binding site of procyanidin B3 on trypsin and pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of procyanidin B3 with trypsin and pepsin and is helpful for understanding its biological activity in vivo.

  11. Trypsin and alpha-chymotrypsin treatment abolishes glibenclamide sensitivity of KATP channels in rat ventricular myocytes.

    PubMed

    Nichols, C G; Lopatin, A N

    1993-03-01

    Cytoplasmic trypsin-treatment of voltage-sensitive potassium channels has been shown to cleave domains of the channel responsible for inactivation of the channel. Trypsin has also been reported to remove slow, irreversible inactivation, or run-down in ATP-sensitive potassium (KATP) channels. Cytoplasmic treatment of rat ventricular KATP channels with either crude, or pure trypsin (1-2 mg/ml) failed to prevent a slow run-down of channel activity. However, trypsin (porcine pancreatic type IX, or type II (Sigma Chem. Co.), or alpha-chymotrypsin (Sigma Chem. Co.) rapidly and irreversibly removed, or substantiallly decreased glibenclamide and tolbutamide-sensitivity of the channels without removing sensitivity to ATP. We conclude that glibenclamide must bind to either a separate protein, or to a separate domain on the channel in order to effect channel inhibition, and this domain is functionally disconnected from the channel by trypsin-, or alpha-chymotrypsin treatment.

  12. Evaluation of the toxicity of ionic liquids on trypsin: A mechanism study.

    PubMed

    Fan, Yunchang; Dong, Xing; Yan, Lingling; Li, Dandan; Hua, Shaofeng; Hu, Chaobing; Pan, Chengcheng

    2016-04-01

    The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative ΔH and ΔS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs.

  13. Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells.

    PubMed

    Clark, Karl J; Carlson, Daniel F; Leaver, Michael J; Foster, Linda K; Fahrenkrug, Scott C

    2009-03-01

    The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

  14. U.S. Geological Survey activities related to American Indians and Alaska Natives: Fiscal year 2004

    USGS Publications Warehouse

    ,; Brunstein, F. Craig

    2006-01-01

    The USGS works in cooperation with American Indian and Alaska Native governments to conduct research on (1) water, energy, and mineral resources, (2) animals and plants that are important for traditional lifeways or have environmental or economic significance, and (3) natural hazards. This report describes most of the activities that the USGS conducted with American Indian and Alaska Native governments, educational institutions, and individuals during Federal fiscal year (FY) 2004. Most of these USGS activities were collaborations with Tribes, Tribal organizations, or professional societies. Other activities were conducted cooperatively with the U.S. Bureau of Indian Affairs (BIA) or other Federal entities.

  15. Does Pedometer Goal Setting Improve Physical Activity among Native Elders? Results from a Randomized Pilot Study

    ERIC Educational Resources Information Center

    Sawchuk, Craig N.; Russo, Joan E.; Charles, Steve; Goldberg, Jack; Forquera, Ralph; Roy-Byrne, Peter; Buchwald, Dedra

    2011-01-01

    We examined if step-count goal setting resulted in increases in physical activity and walking compared to only monitoring step counts with pedometers among American Indian/Alaska Native elders. Outcomes included step counts, self-reported physical activity and well-being, and performance on the 6-minute walk test. Although no significant…

  16. Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

    PubMed

    Merrick, J M; Steger, R; Dombroski, D

    1999-01-01

    Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.

  17. U.S. Geological Survey Activities Related to American Indians and Alaska Natives: Fiscal Year 2005

    USGS Publications Warehouse

    Marcus, Susan M.

    2007-01-01

    Introduction This report describes the activities that the U.S. Geological Survey (USGS) conducted with American Indian and Alaska Native governments, educational institutions, and individuals during Federal fiscal year (FY) 2005. Most of these USGS activities were collaborations with Tribes, Tribal organizations, or professional societies. Others were conducted cooperatively with the Bureau of Indian Affairs (BIA) or other Federal entities. The USGS is the earth and natural science bureau within the U.S. Department of the Interior (DOI). The USGS does not have regulatory or land management responsibilities. As described in this report, there are many USGS activities that are directly relevant to American Indians, Alaska Natives, and to Native lands. A USGS website, dedicated to making USGS more accessible to American Indians, Alaska Natives, their governments, and institutions, is available at www.usgs.gov/indian. This website includes information on how to contact USGS American Indian/Alaska Native Liaisons, training opportunities, and links to other information resources. This report and previous editions are also available through the website. The USGS realizes that Native knowledge and cultural traditions of living in harmony with nature result in unique Native perspectives that enrich USGS studies. USGS seeks to increase the sensitivity and openness of its scientists to the breadth of Native knowledge, expanding the information on which their research is based. USGS scientific studies include data collection, mapping, natural resource modeling, and research projects. These projects typically last 2 or 3 years, although some are parts of longer-term activities. Some projects are funded cooperatively, with USGS funds matched or supplemented by individual Tribal governments, or by the BIA. These projects may also receive funding from the U.S. Environmental Protection Agency (USEPA), the Indian Health Service (part of the Department of Health and Human Services

  18. Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

    PubMed

    Antonini, E; Ascenzi, P; Bolognesi, M; Menegatti, E; Guarneri, M

    1983-04-25

    The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

  19. Hypotonicity activates a native chloride current in Xenopus oocytes

    PubMed Central

    1994-01-01

    Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G- protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium- activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes. PMID:8189203

  20. Anti-tumoral activity of native compound morelloflavone in glioma

    PubMed Central

    Li, Xianfeng; Ai, Hongyan; Sun, Deke; Wu, Tao; He, Jian; Xu, Zhai; Ding, Li; Wang, Ling

    2016-01-01

    The aim of the study was to investigate the anti-tumoral activity of morelloflavone substances with different structures. We also studied the possible link between morelloflavone structure and its function. Various types of chromatographic techniques were used to isolate and screen morelloflavone substances from the extracts of gambogic tree trunk and the morelloflavone structures were identified by analytical techniques such as high resolution mass spectrometry and nuclear magnetism. Anti-tumoral activities of different compounds were investigated and the link between the antitumor activity and the structure of compound was exaimed. Our results showed that the isolated morelloflavone substances demonstrated a certain level of antitumor activity. The compound no. 1 had the strongest effect to inhibit glioma U87 and C6 cells followed by compound no. 2 while compound no. 5 was the weakest among them. We conducted a preliminary analysis on the structure-function relationship through the structure comparison and we concluded that the antitumor effects of morelloflavone substances with different structures were significantly different from each other. Thus, the glucose chain in C4 position of biflavone structure can enhance the antitumor activity of the compound in glioma cells. Additionally, the formation of intramolecular hydrogen bonds in biflavone compounds may also play a role in enhancing the antitumor activity and inhibition rate. PMID:27900007

  1. Antioxidant activity and potential photoprotective from amazon native flora extracts.

    PubMed

    Martins, Francislene J; Caneschi, César A; Vieira, José L F; Barbosa, Wagner; Raposo, Nádia R B

    2016-08-01

    Plant species are sources of active compounds that can fight and/or prevent damage caused by reactive oxygen species, which enables the development of natural products that can help to prevent premature aging caused by exposure to solar radiation. This study assessed the antioxidant and photoprotective activities of six dried extracts of plants from the Brazilian Amazon biome. Plant extracts were prepared in 70% (v/v) ethanol by dynamic maceration for 72h in the dark, and then filtered, concentrated and lyophilized. The extracts were subjected to a phytochemical screening. The antioxidant activity was measured using a 2,2-diphenyl-1-picrylhydrazyl assay and the photoprotection assay was performed using the diffuse transmittance technique. The data obtained from the antioxidant activity assay was evaluated by Student's t-test for independent samples, with the aid of Statistical Package for Social Sciences v.14.0 for Windows software. The flavonoids represent a special metabolites class present in all analyzed extracts. The antioxidant activity (μgmL(-1)) decreased in the following order: Aniba canelilla (1.80±0.16), Brosimum acutifolium (2.84±0.38), Dalbergia monetaria (5.46±0.17) or Caesalpinia pyramidalis (6.45±1.18), Arrabidaea chica (15.35±0.86), and Aspidosperma nitidum (99.14±2.3). Only D. monetaria showed a considerable sun protection factor allowing for labeling (6.0±0.3). The D. monetaria extract was considered the most promising sample because it had optimal antioxidant and photoprotective activities against solar radiation, considering the limit established by regulatory agencies. These extracts with antioxidant potential can be used in photoprotective formulations, providing synergistic photoprotective effect or elevating the adeed value of the product. Additionally, these formulations are attractive to a population who searchs for products made with natural ingredients.

  2. Antibacterial activity of essential oils from Australian native plants.

    PubMed

    Wilkinson, Jenny M; Cavanagh, Heather M A

    2005-07-01

    To date, of the Australian essential oils, only tea tree (Melaleuca alternifolia) and Eucalyptus spp. have undergone extensive investigation. In this study a range of Australian essential oils, including those from Anethole anisata, Callistris glaucophyllia, Melaleuca spp. and Thyptomine calycina, were assayed for in vitro antibacterial activity. M. alternifolia was also included for comparison purposes. Activity was determined using standard disc diffusion assays with each oil assayed at 100%, 10% and 1% against five bacteria (Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Alcaligenes faecalis) and the yeast, Candida albicans. All bacteria, with the exception of Ps. aeruginosa, were susceptible to one or more of the essential oils at 100%, with only Eremophilia mitchelli inhibiting the growth of any bacteria at 1% (inhibition of Sal. typhimurium). Where multiple samples of a single oil variety were tested variability in activity profiles were noted. This suggests that different methods of preparation of essential oils, together with variability in plant chemical profiles has an impact on whether or not the essential oil is of use as an antimicrobial agent. These results show that essential oils from Australian plants may be valuable antimicrobial agents for use alone or incorporated into cosmetics, cleaning agents and pharmaceutical products.

  3. Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

    PubMed

    Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu

    2007-01-01

    SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

  4. Differences in aggression, activity and boldness between native and introduced populations of an invasive crayfish

    USGS Publications Warehouse

    Pintor, L.M.; Sih, A.; Bauer, M.L.

    2008-01-01

    Aggressiveness, along with foraging voracity and boldness, are key behavioral mechanisms underlying the competitive displacement and invasion success of exotic species. However, do aggressiveness, voracity and boldness of the invader depend on the presence of an ecologically similar native competitor in the invaded community? We conducted four behavioral assays to compare aggression, foraging voracity, threat response and boldness to forage under predation risk of multiple populations of exotic signal crayfish Pacifastacus leniusculus across its native and invaded range with and without a native congener, the Shasta crayfish P. fortis. We predicted that signal crayfish from the invaded range and sympatric with a native congener (IRS) should be more aggressive to outcompete a close competitor than populations from the native range (NR) or invaded range and allopatric to a native congener (IRA). Furthermore, we predicted that IRS populations of signal crayfish should be more voracious, but less bold to forage under predation risk since native predators and prey likely possess appropriate behavioral responses to the invader. Contrary to our predictions, results indicated that IRA signal crayfish were more aggressive towards conspecifics and more voracious and active foragers, yet also bolder to forage under predation risk in comparison to NR and IRS populations, which did not differ in behavior. Higher aggression/voracity/ boldness was positively correlated with prey consumption rates, and hence potential impacts on prey. We suggest that the positive correlations between aggression/voracity/boldness are the result of an overall aggression syndrome. Results of stream surveys indicated that IRA streams have significantly lower prey biomass than in IRS streams, which may drive invading signal crayfish to be more aggressive/voracious/bold to acquire resources to establish a population. ?? 2008 The Authors.

  5. Native Americans Today: Resources and Activities for Educators, Grades 4-8.

    ERIC Educational Resources Information Center

    Hirschfelder, Arlene; Beamer, Yvonne

    This activity guide seeks to dispel misrepresentations of Native Americans and build understanding among cultures by offering a hands-on approach to dissecting the whys and hows of institutionalized racism and by painting a realistic and diverse picture of modern American Indians. Each lesson includes a suggested grade level, materials and time…

  6. Online and Face-to-Face Activities of Non-Native English Speakers

    ERIC Educational Resources Information Center

    Winter, Carmen Susanne

    2013-01-01

    The purpose of this study was to examine non-native English speaking students' activity in face-to-face versus online learning environments. The amount of foreign students in the United States increased by 3% in the academic year 2009-2010 (Open Doors, 2010). Adding close to $20 billion to the USA economy, "higher education is among the…

  7. Keepers of the Animals: Native American Stories and Wildlife Activities for Children and Teacher's Guide.

    ERIC Educational Resources Information Center

    Caduto, Michael J.; Bruchac, Joseph

    Twenty-four stories in this book provide a program of study in Native North American Indian culture. The stories introduce the concepts of wildlife ecology and environmental and stewardship issues concerning animals, habitat, and natural history. The field-tested activities encourage creative thinking and synthesis of knowledge and experience by…

  8. Using Active Learning to Teach Culturally Relevant Personal Finance to Native American Students

    ERIC Educational Resources Information Center

    Saboe, Lorna

    2014-01-01

    Active learning is a teaching approach that requires students to do something intellectually with course content. This involves examining, questioning, and relating knowledge gained from previous experiences to new knowledge and skills. Native American students have been found to have low financial literacy skills. Family and consumer sciences…

  9. THE INACTIVATION OF DILUTE SOLUTIONS OF CRYSTALLINE TRYPSIN BY X-RADIATION

    PubMed Central

    McDonald, Margaret R.

    1954-01-01

    The activity of dilute solutions of crystalline trypsin is destroyed by x-rays. The inactivation is an exponential function of the radiation dose. The reaction yield of inactivation is independent of the intensity at which the radiation is delivered or the quality of the x-rays. The reaction yield increases with increasing concentration of trypsin, varying from 0.06 to 0.7 micromoles per liter per 1000 r for trypsin solutions ranging from 1 x 10–7 to 2 x 10–4 M. PMID:13192318

  10. Enzymatic treatment of spermatozoa with a trypsin solution, SpermSolute: improved motility and enhanced ATP concentration.

    PubMed

    Figenschau, Y; Bertheussen, K

    1999-10-01

    We have developed a solution, fully described in this report, that can be used in a pretreatment procedure to promote liquefaction and to enhance motility during preparation of spermatozoa. It was applied to native ejaculates prior to swim-up and, in parallel investigations, motility and adenosine triphosphate concentration were compared in treated and untreated samples, which revealed that the solution significantly improved both parameters. The solution, named SpermSolute, is based on a proteinase (trypsin), which previously has been shown to stimulate the activity of a glycolytic key-enzyme. We speculate that our findings reflect intracellular enzyme activation and we suggest that our formula can be used in sperm preparation to prevent cell aggregates and to promote liquefaction, in addition to promotion of motility.

  11. Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

    PubMed

    Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

    2013-09-15

    Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes.

  12. Diet Overlap and Foraging Activity between Feral Pigs and Native Peccaries in the Pantanal

    PubMed Central

    Galetti, Mauro; Camargo, Hiléia; Siqueira, Tadeu; Keuroghlian, Alexine; Donatti, Camila I.; Jorge, Maria Luisa S. P.; Pedrosa, Felipe; Kanda, Claudia Z.; Ribeiro, Milton C.

    2015-01-01

    Inter-specific competition is considered one of the main selective pressures affecting species distribution and coexistence. Different species vary in the way they forage in order to minimize encounters with their competitors and with their predators. However, it is still poorly known whether and how native species change their foraging behavior in the presence of exotic species, particularly in South America. Here we compare diet overlap of fruits and foraging activity period of two sympatric native ungulates (the white-lipped peccary, Tayassu pecari, and the collared peccary, Pecari tajacu) with the invasive feral pig (Sus scrofa) in the Brazilian Pantanal. We found high diet overlap between white-lipped peccaries and feral pigs, but low overlap between collared peccaries and feral pigs. Furthermore, we found that feral pigs may influence the foraging period of both native peccaries, but in different ways. In the absence of feral pigs, collared peccary activity peaks in the early evening, possibly allowing them to avoid white-lipped peccary activity peaks, which occur in the morning. In the presence of feral pigs, collared peccaries forage mostly in early morning, while white-lipped peccaries forage throughout the day. Our results indicate that collared peccaries may avoid foraging at the same time as white-lipped peccaries. However, they forage during the same periods as feral pigs, with whom they have lower diet overlap. Our study highlights how an exotic species may alter interactions between native species by interfering in their foraging periods. PMID:26536608

  13. 8 CFR 329.5 - Natives of the Philippines with active duty service during World War II.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Natives of the Philippines with active duty service during World War II. 329.5 Section 329.5 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY... Natives of the Philippines with active duty service during World War II. (a) A person desiring...

  14. FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

    PubMed Central

    Kunitz, M.

    1938-01-01

    1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q5–15 = 1.70 to Q25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought

  15. Using Trypsin & Soybean Trypsin Inhibitor to Teach Principles of Enzyme Kinetics

    ERIC Educational Resources Information Center

    Howard, David R.; Herr, Julie; Hollister, Rhiannon

    2006-01-01

    Trypsin and soybean trypsin inhibitor (Kunitz inhibitor) can be used in a relatively simple and inexpensive student exercise to demonstrate the usefulness of enzyme kinetics. The study of enzyme kinetics is essential to biology because enzymes play such a crucial role in the biochemical pathways of all living organisms. The data from enzyme…

  16. Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity.

    PubMed

    Jing, H; Babu, Y S; Moore, D; Kilpatrick, J M; Liu, X Y; Volanakis, J E; Narayana, S V

    1998-10-09

    Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    Purpose 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. Methods 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. Results 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. Conclusions 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

  19. Antioxidant and Anti-Inflammatory Activities of Unexplored Brazilian Native Fruits

    PubMed Central

    Infante, Juliana; Rosalen, Pedro Luiz; Lazarini, Josy Goldoni; Franchin, Marcelo; de Alencar, Severino Matias

    2016-01-01

    Brazilian native fruits are unmatched in their variety, but a poorly explored resource for the development of food and pharmaceutical products. The aim of this study was to evaluate the phenolic composition as well as the antioxidant and anti-inflammatory activities of the extracts of leaves, seeds, and pulp of four Brazilian native fruits (Eugenia leitonii, Eugenia involucrata, Eugenia brasiliensis, and Eugenia myrcianthes). GC—MS analyses of the ethanolic extracts showed the presence of epicatechin and gallic acid as the major compounds in these fruits. Antioxidant activity was measured using synthetic DPPH free-radical scavenging, β-carotene bleaching assay, and reactive oxygen species (ROO·, O2·−, and HOCl). The fruit extracts also exhibited antioxidant effect against biologically relevant radicals such as peroxyl, superoxide, and hypochlorous acid. In general, the pulps were the fruit fractions that exhibited the lowest antioxidant activities, whereas the leaves showed the highest ones. The anti-inflammatory activity was assessed in an in vivo model using the carrageenan-induced neutrophil migration assay, which evaluates the inflammatory response in the acute phase. The pulp, seeds, and leaves of these fruits reduced the neutrophil influx by 40% to 64%. Based on these results, we suggest that the anti-inflammatory activity of these native fruits is related to the modulation of neutrophil migration, through the inhibition of cytokines, chemokines, and adhesion molecules, as well as to the antioxidant action of their ethanolic extracts in scavenging the free-radicals released by neutrophils. Therefore, these native fruits can be useful to produce food additives and functional foods. PMID:27050817

  20. Conversion of protein kinase C from a Ca/sup 2 +/-dependent to an independent form of phorbol ester-binding protein by digestion with trypsin

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-08-29

    Tryptic fragments of protein kinase C containing the kinase (45 KDa) and phorbol ester-binding activity (38 KDa) were separated by Mono O column chromatography. The purified phorbol ester-binding fragment exhibits a higher affinity for phosphatidylserine than the native enzyme but comparable Kd for (/sup 3/H)phorbol 12,13-dibutyrate as the native enzyme. This proteolytic fragment binds phorbol ester equally efficient either in the presence or absence of Ca/sup 2 +/ and the addition of the kinase fragment did not restore the Ca/sup 2 +/-requirement for the binding. These results indicate that protein kinase C is composed of two functionally distinct units which can be expressed independently after limited proteolysis with trypsin.

  1. Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

    PubMed Central

    Zhou, Dongwen; Lobo, Yara A.; Batista, Isabel F. C.; Marques-Porto, Rafael; Gustchina, Alla; Oliva, Maria L. V.; Wlodawer, Alexander

    2013-01-01

    A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes. PMID:23626794

  2. Two crystal structures of the leupeptin-trypsin complex.

    PubMed Central

    Kurinov, I. V.; Harrison, R. W.

    1996-01-01

    Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57. PMID:8845765

  3. Stability and kinetics of a bifunctional amylase/trypsin inhibitor.

    PubMed

    Alagiri, S; Singh, T P

    1993-11-10

    The stability of the bifunctional amylase/trypsin inhibitor from ragi (Indian finger millet, Eleusine coracana) has been studied by methods of circular dichroism, UV absorption and intrinsic fluorescence. The inhibitor is stable in 8 M urea and 6 M guanidine-HCl. In 150 mM NaCl, thermal denaturation does not occur up to 90 degrees C. However, it is irreversibly denatured in 5 mM NaCl if heated over 73 degrees C. The acidic denaturation is reversible in both high and low salt conditions, but it shows different behavior below pH 1.65 under similar salt conditions. The helical content is about 2-4% in the pH range of 7-9 at which the inhibitor is active maximally. The NaCl concentration does not have a significant effect on the secondary structure elements. The beta-strand form does not show much variation under various conditions. Arg34-Leu35 is the reactive peptide bond in the trypsin-binding site. Trp and Tyr are involved in the binding with amylase. The bifunctional inhibitor represents the sum of individual inhibitors of trypsin and amylase.

  4. Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme.

    PubMed

    Schroeder, L L; Pappas, P W; Means, G E

    1981-06-01

    In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.

  5. Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for trypsin digestion column

    SciTech Connect

    Jun, Seung-Hyun; Chang, Mun Seock; Kim, Byoung Chan; An, Hyo Jin; Lopez-Ferrer, Daniel; Zhao, Rui; Smith, Richard D.; Lee, Sang-Won; Kim, Jungbae

    2010-09-15

    The construction of a trypsin reactor in a chromatography column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coating, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on asspun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the enzymes were improved yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coating showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin. The combination of highly stable trypsin coating and alcoholdispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.

  6. Comparing citrated native, kaolin-activated, and tissue factor-activated samples and determining intraindividual variability for feline thromboelastography.

    PubMed

    Banerjee, Amrita; Blois, Shauna L; Wood, R Darren

    2011-11-01

    Thromboelastography (TEG) is a point-of-care whole blood test of hemostasis. While TEG is becoming more widely used in veterinary medicine, few studies describe the use of TEG in cats. The objectives of the current study were to: 1) document the range of TEG variables produced in healthy cats using 3 sample types (citrated native, kaolin-activated, and tissue factor-activated), and 2) determine if there was a significant difference between 2 separate samples obtained from individual healthy cats on the same day. Jugular venipuncture was performed in 20 cats, and citrated blood collected for TEG. TEG analysis was performed on citrated native, kaolin-activated, and tissue factor-activated blood for each sample. Two hours later, the procedure was repeated from the opposite jugular vein, yielding a total of 120 analyses. Reaction time (R), alpha angle (α), kappa value (κ), and maximum amplitude (MA) were recorded from each tracing. No significant differences were found between TEG tracings from the first and second venipuncture samples. Significant differences were found between sample types for R, α, κ, and MA. Means for citrated native/kaolin-activated/tissue factor-activated methods were R = 4.1/3.7/0.6 min; κ = 2.5/1.8/2.2 min; α = 59.9/65.1/70.4 degrees; MA = 47.4/49.9/44.7 mm. A limitation of this study was the small number of cats used. Thromboelastography analysis may be a suitable method of evaluating hemostasis in cats.

  7. Crystal structure of an extensively simplified variant of bovine pancreatic trypsin inhibitor in which over one-third of the residues are alanines.

    PubMed

    Islam, Mohammad Monirul; Sohya, Shihori; Noguchi, Keiichi; Yohda, Masafumi; Kuroda, Yutaka

    2008-10-07

    We report the high-resolution crystal structures of an extensively simplified variant of bovine pancreatic trypsin inhibitor containing 20 alanines (BPTI-20st) and a reference single-disulfide-bonded variant (BPTI-[5,55]st) at, respectively, 1.39 and 1.09 A resolutions. The sequence was simplified based on the results of an alanine scanning experiment, as reported previously. The effects of the multiple alanine substitutions on the overall backbone structure were surprisingly small (C(alpha) atom RMSD of 0.53 A) being limited to small local structural perturbations. Both BPTI variants retained a wild-type level of trypsin inhibitory activity. The side-chain configurations of residues buried in the hydrophobic cores (<30% accessible surface area) were almost perfectly retained in both BPTI-20st and BPTI-[5,55]st, indicating that neither multiple alanine replacements nor the removal of the disulfide bonds affected their precise placements. However, the side chains of three partially buried residues (Q31, R20, and to some extent Y21) and several unburied residues rearranged into alternative dense-packing structures, suggesting some plasticity in their shape complementarity. These results indicate that a protein sequence simplified over its entire length can retain its densely packed, native side-chain structure, and suggest that both the design and fold recognition of natively folded proteins may be easier than previously thought.

  8. Toxic interaction between acid yellow 23 and trypsin: spectroscopic methods coupled with molecular docking.

    PubMed

    Wang, Jing; Liu, Rutao; Qin, Pengfei

    2012-09-01

    Acid yellow 23 (AY23) is a pervasive azo dye used in many fields which is potentially harmful to the environment and human health. This paper studied the toxic effects of AY23 on trypsin by spectroscopic and molecular docking methods. The addition of AY23 effectively quenched the intrinsic fluorescence of trypsin via static quenching with association constants of K(290 K) = 3.67 × 10(5) L mol(-1) and K(310 K) = 1.83 × 10(5) L mol(-1). The calculated thermodynamic parameters conformed that AY23 binds to trypsin predominantly via electrostatic forces with one binding site. Conformational investigations indicated the skeletal structure of trypsin unfolded and the microenvironment of tryptophan changed with the addition of AY23. Molecular docking study showed that AY23 interacted with the His 57 and Lys 224 residue of trypsin and led to the inhibition of enzyme activity. This study offers a more comprehensive picture of AY23-trypsin interaction and indicates their interaction may perform toxic effects within the organism.

  9. Inhibitory effects of daidzein and genistein on trypsin: Insights from spectroscopic and molecular docking studies.

    PubMed

    Zeng, Hua-Jin; Wang, Ya-Ping; Yang, Ran; You, Jing; Qu, Ling-Bo

    2016-08-01

    In this work, the inhibitory effect of two isoflavonoids including daidzein and genistein on trypsin and their binding mechanism were determined by spectroscopic and molecular docking approaches. The results indicated that both daidzein and genistein reversibly inhibited trypsin in a competitive manner with IC50 values of 68.01×10(-6)molL(-1) and 64.70×10(-6)molL(-1) and Ki values of 62.12×10(-6)molL(-1) and 59.83×10(-6)molL(-1), respectively. They could spontaneously bind with trypsin mainly through hydrophobic force and electrostatic interactions with a single binding site. Analysis of circular dichrosim spectra and molecular docking revealed that both isoflavonoids bound directly into the catalytic cavity and the microenvironment and secondary structure of trypsin were changed in this process, which caused the inhibition of trypsin activity. All these experimental results and theoretical data in this work would be help in understanding the mechanism of inhibitory effects of daidzein and genistein against trypsin and the potential of isoflavonoid to relieve symptoms of pancreatitis.

  10. Computational analysis of the MCoTI-II plant defence knottin reveals a novel intermediate conformation that facilitates trypsin binding

    PubMed Central

    Jones, Peter M.; George, Anthony M.

    2016-01-01

    MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors’ dynamics that may be of utility in drug design. PMID:26975976

  11. Computational analysis of the MCoTI-II plant defence knottin reveals a novel intermediate conformation that facilitates trypsin binding

    NASA Astrophysics Data System (ADS)

    Jones, Peter M.; George, Anthony M.

    2016-03-01

    MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors’ dynamics that may be of utility in drug design.

  12. Computational analysis of the MCoTI-II plant defence knottin reveals a novel intermediate conformation that facilitates trypsin binding.

    PubMed

    Jones, Peter M; George, Anthony M

    2016-03-15

    MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors' dynamics that may be of utility in drug design.

  13. Ectopic trypsin in the myocardium promotes dilated cardiomyopathy after influenza A virus infection.

    PubMed

    Pan, Hai-Yan; Sun, Hua-Mei; Xue, Lu-Jing; Pan, Min; Wang, Yi-Ping; Kido, Hiroshi; Zhu, Jian-Hua

    2014-09-15

    We have previously reported that ectopic trypsin in the myocardium triggers acute myocarditis after influenza A virus (IAV) infection. As myocarditis is a common precursor to dilated cardiomyopathy (DCM), the aim of the present study was to investigate the influence of trypsin on the progression of DCM after IAV infection. IAV-infected mice treated with saline or trypsin inhibitor were euthanized on days 0, 9, 20, 40 and 60 postinfection. Trypsin expression colocalized with myocardial inflammatory loci and IAV-induced myocarditis peaked on day 9 postinfection and alleviated by day 20 but persisted until day 60 postinfection, even though replication of IAV was not detected from day 20 postinfection. Similar time courses were observed for the activation of pro-matrix metalloproteinase (pro-MMP)-9 and expression of the proinflammatory cytokines IL-6, IL-1β, and TNF-α. Degradation of collagen type I, proliferation of ventricular interstitial collagen, and expression of collagen type I and III mRNA increased significantly during acute and chronic phases; collagen type III mRNA increased more significantly than collagen type I mRNA. Cardiac function progressively deteriorated with progressive left ventricular dilation. The trypsin inhibitor aprotinin suppressed pro-MMP-9 activation and cytokine release, alleviated myocardial inflammation, and restored collagen metabolism during acute and chronic phases of myocarditis. This effectively prevented ventricular dilation and improved cardiac function. These results suggest that ectopic trypsin in the myocardium promoted DCM through chronic activation of pro-MMP-9, persistent induction of cytokines, and mediation of collagen remodeling. Pharmacological inhibition of trypsin activity might be a promising approach for the prevention of viral cardiomyopathy.

  14. Inactivation of trypsin inhibitors in sweet potato and taro tubers during processing.

    PubMed

    Kiran, K Sasi; Padmaja, G

    2003-01-01

    In order to understand the extent of elimination of trypsin inhibitors during processing of sweet potato (Ipomoea batatas) and taro (Colocasia esculenta) tubers, a detailed study was conducted using tubers processed by oven drying, cooking, and microwave baking. Between 80 and 90% trypsin inhibitor (TI) activity was retained in sweet potato chips up to 2h at 70 degrees C. Among the four cultivars of sweet potatoes, RS-III-2 trypsin inhibitors were more heat labile. Heating at 100 degrees C led to rapid inactivation of TI of sweet potatoes. Varietal differences in thermal stability were more pronounced for the trypsin inhibitors of taro than sweet potatoes. Taro inhibitors were also more rapidly inactivated than sweet potato TI. Between 17 and 31% TI activity was retained in cooked tuber pieces of sweet potatoes, while only 3-10% were retained in taro cultivars. Very effective inactivation of trypsin inhibitors of sweet potatoes and taro could be obtained through microwave baking. Flour prepared from taro was devoid of TI activity, while 5-12% TI activity was retained in the flour prepared from sweet potatoes. The study clearly established that among the four techniques used, microwave baking and flour preparation were the best methods to eliminate TI from sweet potatoes and taro.

  15. The influence of parental nativity, neighborhood disadvantage and the built environment on physical activity behaviors in Latino youth.

    PubMed

    Echeverría, Sandra E; Ohri-Vachaspati, Punam; Yedidia, Michael J

    2015-04-01

    Little evidence exists examining if parental nativity, neighborhood disadvantage and built environment features are associated with physical activity behaviors in Latino youth. We used a representative sample of Latino youth (n = 616) living in New Jersey to examine parental nativity associations with active transport to school, active use of sidewalks, use of local neighborhood parks, and use of neighborhood physical activity facilities. We estimated prevalence ratios (PR) that accounted for the complex survey design. Latino youth with foreign-born parents were generally more active than their US-born peers, and those with parents in the US 10 years or less were more likely to engage in active transport to school (PR = 1.51, 95% CI 1.04-2.21), after adjusting for census-based neighborhood disadvantage, self-reported neighborhood measures, and geocoded distance to school. Parental nativity status should be considered in policies or interventions designed to increase physical activity among Latino youth.

  16. The Influence of Parental Nativity, Neighborhood Disadvantage and the Built Environment on Physical Activity Behaviors in Latino Youth

    PubMed Central

    Ohri-Vachaspati, Punam; Yedidia, Michael J.

    2016-01-01

    Little evidence exists examining if parental nativity, neighborhood disadvantage and built environment features are associated with physical activity behaviors in Latino youth. We used a representative sample of Latino youth (n = 616) living in New Jersey to examine parental nativity associations with active transport to school, active use of sidewalks, use of local neighborhood parks, and use of neighborhood physical activity facilities. We estimated prevalence ratios (PR) that accounted for the complex survey design. Latino youth with foreign-born parents were generally more active than their US-born peers, and those with parents in the US 10 years or less were more likely to engage in active transport to school (PR = 1.51, 95 % CI 1.04–2.21), after adjusting for census-based neighborhood disadvantage, self-reported neighborhood measures, and geocoded distance to school. Parental nativity status should be considered in policies or interventions designed to increase physical activity among Latino youth. PMID:24162884

  17. Human β-defensin 4 with non-native disulfide bridges exhibit antimicrobial activity.

    PubMed

    Sharma, Himanshu; Nagaraj, Ramakrishnan

    2015-01-01

    Human defensins play multiple roles in innate immunity including direct antimicrobial killing and immunomodulatory activity. They have three disulfide bridges which contribute to the stability of three anti-parallel β-strands. The exact role of disulfide bridges and canonical β-structure in the antimicrobial action is not yet fully understood. In this study, we have explored the antimicrobial activity of human β-defensin 4 (HBD4) analogs that differ in the number and connectivity of disulfide bridges. The cysteine framework was similar to the disulfide bridges present in μ-conotoxins, an unrelated class of peptide toxins. All the analogs possessed enhanced antimicrobial potency as compared to native HBD4. Among the analogs, the single disulfide bridged peptide showed maximum potency. However, there were no marked differences in the secondary structure of the analogs. Subtle variations were observed in the localization and membrane interaction of the analogs with bacteria and Candida albicans, suggesting a role for disulfide bridges in modulating their antimicrobial action. All analogs accumulated in the cytosol where they can bind to anionic molecules such as nucleic acids which would affect several cellular processes leading to cell death. Our study strongly suggests that native disulfide bridges or the canonical β-strands in defensins have not evolved for maximal activity but they play important roles in determining their antimicrobial potency.

  18. Unraveling the contributions of hydrogen-bonding interactions to the activity of native and non-native ligands in the quorum-sensing receptor LasR

    PubMed Central

    Gerdt, Joseph P.; McInnis, Christine E.; Schell, Trevor L.; Blackwell, Helen E.

    2014-01-01

    Quorum sensing (QS) via the synthesis and detection of N-acyl L-homoserine lactone (AHL) signals regulates important pathogenic and mutualistic phenotypes in many bacteria. Over the past two decades, the development of non-native molecules that modulate this cell-cell signaling process has become an active area of research. The majority of these compounds were designed for block binding of the native AHL signal to its cognate LuxR-type receptor, and much effort has focused on LasR in the opportunistic pathogen Pseudomonas aeruginosa. Despite a small set of reported LasR structural data, it remains unclear which polar interactions are most important for either (i) activation of the LasR receptor by its native AHL signal, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL), or (ii) activation or inhibition of LasR by related AHL analogs. Herein, we report our investigations into the activity of OdDHL and five synthetic analogs in wild-type LasR and in nine LasR mutants with modifications to key polar residues in their ligand binding sites. Our results allowed us to rank, for the first time, the relative importance of each LasR:OdDHL hydrogen bond for LasR activation and provide strong evidence for the five synthetic ligands binding LasR in a very similar orientation as OdDHL. By delineating the specific molecular interactions that are important for LasR modulation by AHLs, these findings should aid in the design of new synthetic modulators of LasR (and homologous LuxR-type receptors) with improved potencies and selectivities. PMID:25474181

  19. Unexpected Trypsin Cleavage at Ubiquitinated Lysines

    PubMed Central

    2015-01-01

    Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers—linear Ub–48Ub–48Ub, linear Ub–63Ub–63Ub, and the branched trimer [Ub]2–6,48Ub—have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin. PMID:26182167

  20. Antioxidant Activities and Phytochemicals of Leaf Extracts from 10 Native Rhododendron Species in Taiwan

    PubMed Central

    Lin, Chi-Yang; Lin, Lei-Chen; Ho, Shang-Tse; Tung, Yu-Tang; Tseng, Yen-Hsueh

    2014-01-01

    Rhododendron, one of the most famous ornamental plants in the world, is traditionally a medicinal plant. However, the potential bioactivities of native Rhododendron in Taiwan have not been completely studied. In this study, the results revealed that Rhododendron pseudochrysanthum exhibited the best antioxidant activities among 10 native Rhododendron species in Taiwan. Furthermore, based on a bioactivity-guided isolation principle, nine specific phytochemicals were isolated and identified as (2R,3S)-catechin (1), (2R,3R)-epicatechin (1′), (2R,3R)-dihydromyricetin 3-O-β-l-arabinopyranoside (2), (2S,3S)-taxifolin 3-O-β-l-arabinopyranoside (2′), (2R,3R)-taxifolin 3-O-β-l-arabinopyranoside (3), myricetin 3-O-β-d-glucopyranoside (3′), rutin (4), hyperoside (5), and quercitrin (6). Of these compounds, 2 and 3 were found to be major bioactive compounds, and their concentrations in the n-butanol (BuOH) fraction were determined to be 52.0 and 67.3 mg per gram, respectively. These results demonstrated that methanolic extracts of Rhododendron pseudochrysanthum leaves have excellent antioxidant activities and great potential as a source for natural health products. PMID:24987425

  1. Isolation of a trypsin-chymotrypsin inhibitor and its functional properties.

    PubMed

    Wang, Shaoyun; Shao, Biao; Lu, Wei; Hong, Jing; Rao, Pingfan

    2014-01-01

    A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman-Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85 °C, as well as within the pH range 2-12. Besides inhibiting trypsin-chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.

  2. Trypsin-catalyzed multicomponent reaction: A novel and efficient one-pot synthesis of thiazole-2-imine derivatives.

    PubMed

    Zhou, Junbin; Huang, Xingtian; Zhang, Zhuan; Song, Ping; Li, Yiqun

    2017-01-10

    The first Trypsin from porcine pancreas catalyzed a novel one-pot three-component reaction of α-bromoketone, primary alkylamines, and phenylisothiocyanate for the synthesis of thiazole-imine derivatives with high yields (up to 98%) in a short time under mild conditions. The results revealed that Trypsin exhibited excellent catalytic activity and great tolerance for broad substrates. This Trypsin-catalyzed three component convergent method provides a novel strategy for the synthesis of thiazole-2-imine derivatives and expands the promiscuous functions of enzymes in organic synthesis.

  3. Effects of organic solvents and substrate binding on trypsin in acetonitrile and hexane media.

    PubMed

    Meng, Yanyan; Yuan, Yuan; Zhu, Yanyan; Guo, Yanzhi; Li, Menglong; Wang, Zhimeng; Pu, Xuemei; Jiang, Lin

    2013-09-01

    In this work, we used molecular dynamic (MD) simulation to study trypsin with and without a six-amino-acid peptide bound in three different solvents (water, acetonitrile and hexane) in order to provide molecular information for well understanding the structure and function of enzymes in non-aqueous media. The results show that the enzyme is more compact and less native-like in hexane than in the other two polar solvents. The substrate could stabilize the native protein structure in the two polar media, but not in the non-polar hexane. There are no significant differences in the conformation of the S1 pocket upon the substrate binding in water and acetonitrile media while a reverse behavior is observed in hexane media, implying a possible induced fit binding mechanism in the non-polar media. The substrate binding enhances the stability of catalytic H-bond network since it could expel the solvent molecules from the active site. The enzyme and the substrate appear to be more appropriate to the reactive conformation in the organic solvents compared with aqueous solution. There is much greater substrate binding strength in hexane media than the water and acetonitrile ones since the polar solvent significantly weakens electrostatic interactions, which are observed to be the main driving force to the binding. In addition, some residues of the S1 pocket could remain favorable contribution to the binding despite the solvent change, but with differences in the contribution extent, the number and the type of residues between the three media.

  4. Increased lung vascular permeability after pancreatitis and trypsin infusion.

    PubMed Central

    Tahamont, M. V.; Barie, P. S.; Blumenstock, F. A.; Hussain, M. H.; Malik, A. B.

    1982-01-01

    We examined the role of proteases in mediating lung vascular injury after acute hemorrhagic pancreatitis. Studies were made in sheep in which pulmonary lymph was collected for assessment of the changes in transvascular fluid and protein exchange. The induction of pancreatitis by injection of trypsin and sodium taurocholate into the pancreas resulted in increases in pulmonary lymph flow and transvascular protein clearance (lymph flow x lymph-to-plasma protein concentration ratio). The pulmonary vascular pressures did not change significantly after pancreatitis, indicating that the increases in pulmonary lymph flow and protein clearance were due to increased pulmonary endothelial permeability. The response to pancreatitis was also characterized by decreases in concentrations of fibrinogen, platelets, and granulocytes. Pulmonary leukostasis was a common morphologic feature in this group. In another group, an intravenous infusion of trypsin, which produced decreases in antiprotease activity comparable to those observed after pancreatitis, also resulted in increases in pulmonary lymph flow and transvascular protein clearance. These increases in lymph fluxes were comparable to those observed after pancreatitis and were also associated with decreases in concentrations of fibrinogen, platelets, and granulocytes. Pulmonary leukostasis was evident in this group upon histologic examination. In a third group, pretreatment with Trasylol prevented the increases in pulmonary lymph flow and transvascular protein clearance after pancreatitis, suggesting that the pancreatitis-induced pulmonary vascular injury is the result of the release of proteases. The results indicate a common pulmonary vascular response to acute pancreatitis and trypsin infusion. The release of proteases into the circulation after acute pancreatitis may be the initiating event mediating the pulmonary vascular injury. Images Figure 7 Figure 8 Figure 9 Figure 10 Figures 11 and 12 PMID:6181692

  5. A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

    PubMed

    Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

    2014-01-01

    A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

  6. Biological activity of acyl glucose esters from Datura wrightii glandular trichomes against three native insect herbivores.

    PubMed

    Hare, J Daniel

    2005-07-01

    Datura wrightii is dimorphic for leaf trichome type in southern California. "Sticky" plants produce glandular trichomes that secrete acylsugars, whereas velvety plants produce nonglandular trichomes. Glandular trichomes confer resistance to some potential insect herbivores and are associated with reduced feeding in the field by two native coleopteran herbivores: the tobacco flea beetle, Epitrix hirtipennis, and a weevil, Trichobaris compacta. In contrast, another native beetle, Lema daturaphila, damages sticky and velvety plants similarly in the field. A series of choice and no-choice "ester removal" and "ester addition" feeding experiments were performed in the laboratory to evaluate the role of acylsugars in feeding by all three insect species. Consumption of sticky leaves after their esters were removed by washing was compared to consumption of unwashed sticky leaves and velvety leaves in ester removal experiments. Consumption of velvety leaves was measured after acylsugars were applied to those leaves in controlled amounts in the ester addition experiments. Consumption by E. hirtipennis was reduced by acylsugars in all experiments. Consumption by T. compacta was reduced by acylsugars in the ester removal experiments, but not in the ester addition experiments. The location of the acylsugars at the tip of a long trichome, rather than simply on the leaf surface, may be an important component of the biological activity of acylsugars against T. compacta in nature. Consumption by L. daturaphila was not significantly reduced by acylsugars in any experiment. The acylsugars caused no significant mortality of any of the three insect species.

  7. Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

    PubMed Central

    Cui, Fengchao; Yang, Kecheng; Li, Yunqi

    2015-01-01

    To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas. PMID:25938485

  8. Detecting trypsin at liquid crystal/aqueous interface by using surface-immobilized bovine serum albumin.

    PubMed

    Chuang, Cheng-Hao; Lin, Yi-Cheng; Chen, Wei-Long; Chen, Yu-Hsuan; Chen, Yu-Xun; Chen, Chieh-Ming; Shiu, Hung Wei; Chang, Lo-Yueh; Chen, Chia-Hao; Chen, Chih-Hsin

    2016-04-15

    We report a new mechanism for liquid crystal (LC)-based sensor system for trypsin detection. In this system, bovine serum albumin (BSA) was immobilized on gold grids as the enzymatic substrate. When the BSA-modified grid was filled with LC and immersed in the solution containing trypsin, the peptide bonds of BSA were hydrolyzed and peptide fragments were desorbed from the surface of gold grid, which disrupted the orientation of LC at the vicinity and resulted in a dark-to-bright transition of optical image of LCs. By using this mechanism, the limit of detection (LOD) of trypsin is 10 ng/mL, and it does not respond to thrombin and pepsin. Besides, the cleavage behavior on gold surfaces was directly visualized by the scanning photoelectron microscopy (SPEM), in particular for the chemical composition identification and element-resolved image. The loss of BSA fragments and the enhancement of Au photoelectron signal after trypsin cleavage were corresponding to the proposed mechanism of the LC-based sensor system. Because the signals reported by LC can be simply interpreted through the human naked-eye, it provides a simple method for fast-screening trypsin activity in aqueous solution.

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

    SciTech Connect

    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.

  10. STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

    PubMed

    Huang, Guo N; Zeng, Weizhong; Kim, Joo Young; Yuan, Joseph P; Han, Linhuang; Muallem, Shmuel; Worley, Paul F

    2006-09-01

    Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

  11. Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue.

    PubMed

    Del Lungo, Martina; Melchiorre, Michele; Guandalini, Luca; Sartiani, Laura; Mugelli, Alessandro; Koncz, Istvan; Szel, Tamas; Varro, Andras; Romanelli, Maria Novella; Cerbai, Elisabetta

    2012-05-01

    BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f) by 67% at -120 mV, while MEL57A (3 µM) reduced I(f) by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.

  12. Cytoprotective and pro-apoptotic activities of native Australian herbs polyphenolic-rich extracts.

    PubMed

    Sakulnarmrat, Karunrat; Fenech, Michael; Thomas, Philip; Konczak, Izabela

    2013-01-01

    Three commercially grown native herbs unique to Australia, Tasmannia pepper leaf (Tasmannia lanceolata R. Br., Winteracea; TPL), anise myrtle (Syzygium anisatum Vickery, Craven & Biffen, Myrtaceae; AM) and lemon myrtle (Backhousia citriodora F. Muell, Myrtaceae; LM) as well as a reference sample bay leaf (Laurus nobilis L., Lauraceae; BL) were examined for potential cytoprotective properties. All native herbs exhibited greater cellular antioxidant activity as measured by the cellular antioxidant activity (CAA) assay than bay leaf and reduced the hydrogen peroxide (H(2)O(2)) induced death of hepatocellular carcinoma (HepG2) cells by 25-50%. All herb extracts reduced the proliferation of colon (HT-29; IC(50)=0.75-1.39mg/ml), stomach (AGS; IC(50)=0.59-1.88mg/ml), bladder (BL13; IC(50)=0.56-1.12mg/ml) and liver (HepG2; IC(50)=0.38-1.36mg/ml) cancer cells. No significant reduction of cell viability of non-transformed colon (CCD-18Co; IC(50)>2.0mg/ml) and mixed stomach and intestine (Hs 738.St/Int; IC(50)>2.0mg/ml) cells was observed. Flow cytometry analysis and the results of the cytokinesis block micronucleus cytome (CBMNCyt) assay conducted with respectively, promyelocytic leukaemia (HL-60) and colon adenocarcinoma (HT-29) cells suggest an increase in apoptosis following treatment with the herb extracts. The occurrence of apoptotic cells coincided with an increase in caspase-3 enzyme activity. The results of the CBMNCyt assay suggested no direct DNA damage in colon adenocarcinoma (HT-29) cells as a result of treatment with all extracts, applied at final concentrations of 0.5 and 1.0mg/ml.

  13. Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification.

    PubMed

    Jónsdóttir, Gudrún; Bjarnason, Jón Bragi; Gudmundsdóttir, Agústa

    2004-01-01

    Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.

  14. Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

    PubMed Central

    Ikeda, Keiko; Steger, David J.; Eberharter, Anton; Workman, Jerry L.

    1999-01-01

    Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions. PMID:9858608

  15. Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L. 1

    PubMed Central

    Metzger, James D.

    1990-01-01

    Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A1, (GA1), GA9, and GA20 caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA53, or GA8. Moreover, of the biologically active compounds, GA9 was the most active followed closely by GA1. In thermoinduced plants (4-week cold treatment at 6°C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA9 was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA20. No such increase in activity was observed for either GA[unk] or GA53. The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA9 is the endogenous mediator of thermoinduced stem growth in field pennycress. PMID:16667681

  16. Superior temporal activation as a function of linguistic knowledge: insights from deaf native signers who speechread.

    PubMed

    Capek, Cheryl M; Woll, Bencie; MacSweeney, Mairéad; Waters, Dafydd; McGuire, Philip K; David, Anthony S; Brammer, Michael J; Campbell, Ruth

    2010-02-01

    Studies of spoken and signed language processing reliably show involvement of the posterior superior temporal cortex. This region is also reliably activated by observation of meaningless oral and manual actions. In this study we directly compared the extent to which activation in posterior superior temporal cortex is modulated by linguistic knowledge irrespective of differences in language form. We used a novel cross-linguistic approach in two groups of volunteers who differed in their language experience. Using fMRI, we compared deaf native signers of British Sign Language (BSL), who were also proficient speechreaders of English (i.e., two languages) with hearing people who could speechread English, but knew no BSL (i.e., one language). Both groups were presented with BSL signs and silently spoken English words, and were required to respond to a signed or spoken target. The interaction of group and condition revealed activation in the superior temporal cortex, bilaterally, focused in the posterior superior temporal gyri (pSTG, BA 42/22). In hearing people, these regions were activated more by speech than by sign, but in deaf respondents they showed similar levels of activation for both language forms - suggesting that posterior superior temporal regions are highly sensitive to language knowledge irrespective of the mode of delivery of the stimulus material.

  17. Antioxidant and Antimycotic Activities of Two Native Lavandula Species from Portugal

    PubMed Central

    Baptista, Rafael; Madureira, Ana Margarida; Jorge, Rita; Adão, Rita; Duarte, Aida; Duarte, Noélia; Lopes, Maria Manuel; Teixeira, Generosa

    2015-01-01

    The antioxidant and antimycotic activities of the essential oils and extracts of two native Portuguese Lavandula species, L. stoechas subsp. luisieri and L. pedunculata, were evaluated by in vitro assays. The total phenolics and flavonoids content were also determined. The antioxidant potential was assessed through DPPH radical scavenging, inhibition of lipid peroxidation (ILP), and DNA protection assays. All samples displayed a high DPPH scavenging activity, some of them showing concentration dependence. The majority of the samples were also able to inhibit lipid peroxidation. A strong correlation was observed between the results of DPPH and ILP assays and the flavonoids content of the samples. In the DNA protection assay, all the extracts were able to preserve DNA integrity. The antimycotic activity was performed against twelve fungi belonging to Basidiomycota and Ascomycota Divisions. L. stoechas subsp. luisieri exhibited the broadest activity spectra. L. pedunculata extracts were active against five fungi. Cryptococcus neoformans was the most sensitive, being inhibited by all the extracts. Our results led to the conclusion that L. stoechas subsp. luisieri and L. pedunculata can be useful as new sources of natural antioxidants and antimycotic agents, providing a possible valorization of the existing biodiversity and resources of Portuguese flora. PMID:25922611

  18. Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV.

    PubMed

    Karle, Michael; Knecht, Wolfgang; Xue, Yafeng

    2012-07-15

    In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.

  19. Mechanism of curcumin-induced trypsin inhibition: Computational and experimental studies

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Qing; Zhang, Hong-Mei; Kang, Yi-Jun; Gu, Yun-Lan; Cao, Jian

    2016-03-01

    In the present study, the experimental and theoretical methods were used to analyze the binding interaction of food dye, curcumin with trypsin. The results of fluorescence spectroscopic measurements indicated that curcumin binding resulted in the obviously intrinsic fluorescence quenching with the increase concentration of curcumin. This binding interaction is a spontaneous process with the estimated enthalpy and entropy changes being -15.70 kJ mol-1 and 40.25 J mol-1 K-1, respectively. Hydrogen bonds and hydrophobic forces played an important role in the complex formation between curcumin and trypsin. Moreover, curcumin could enter into the primary substrate-binding pocket and makes the activity of trypsin decrease remarkably with the increasing concentration of curcumin.

  20. 8 CFR 329.5 - Natives of the Philippines with active duty service during World War II.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Natives of the Philippines with active duty service during World War II. 329.5 Section 329.5 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY... of the Philippines with active duty service during World War II. (a) A person desiring to...

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

    PubMed

    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.

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26694369

  3. Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Proactive action to examine trypsin gene profiles and proteinase inhibitors for interaction with Bt toxin is necessary ...

  4. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity

    PubMed Central

    Kaltenbach, Miriam; Emond, Stephane; Tokuriki, Nobuhiko

    2016-01-01

    The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with “evolvability” was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and

  5. Review of techniques to prevent introduction of zebra mussels (Dreissena polymorpha) during native mussel (Unionoidea) conservation activities

    USGS Publications Warehouse

    Cope, W.G.; Newton, T.J.; Gatenby, C.M.

    2003-01-01

    Because of the declines in diversity and abundance of native freshwater mussels (superfamily Unionoidea), and the potential decimation of populations of native mussels resulting from the rapid spread of the exotic zebra mussel Dreissena polymorpha, management options to eliminate or reduce the threat of the zebra mussel are needed. Relocating native mussels to refugia (artificial and natural) has been proposed to mitigate the threat of zebra mussels to native species. Relocation of native mussels to refugia such as fish hatchery facilities or natural habitats within their historic range. Which are unlikely to be infested by zebra mussels, necessitates that protocols be developed to prevent the inadvertent introduction of zebra mussels. Several recent studies have developed Such protocols, and have assessed their effectiveness on the health and survival of native mussels during subsequent relocation to various refugia. The purpose of this project is to synthesize and evaluate the current protocols and to develop a set of procedures that resource managers and researchers should consider before conducting conservation activities in zebra mussel infested waters. We found that the existing protocols have many common points of concern, such as facility modification and suitability, zebra mussel risk assessment and management procedures, and health and disease management procedures. These conservation protocols may have broad applicability to other situations and locations. A summary and evaluation of the information in these main areas, along with recommended guidelines, are presented in this article.

  6. Antioxidant Enzyme Activity, Iron Content and Lipid Oxidation of Raw and Cooked Meat of Korean Native Chickens and Other Poultry

    PubMed Central

    Muhlisin; Utama, Dicky Tri; Lee, Jae Ho; Choi, Ji Hye; Lee, Sung Ki

    2016-01-01

    This study was conducted to observe antioxidant enzyme activity, iron content and lipid oxidation of Korean native chickens and other poultry. The breast and thigh meat of three Korean native chicken breeds including Woorimatdak, Hyunin black and Yeonsan ogye, and three commercial poultry breeds including the broiler, White Leghorn and Pekin duck (Anasplatyrhyncos domesticus) were studied. The analyses of the antioxidant enzymes activity, iron content and lipid oxidation were performed in raw and cooked samples. The activity of catalase (CAT) in the thigh meat was higher than that of the breast meat of three Korean native chickens and the broiler, respectively. The activity of glutathione peroxidase (GPx) in the uncooked thigh meat of three Korean native chickens was higher than that of the breasts. The breast meat of Woorimatdak and Pekin duck had higher superoxide dismutase (SOD) activity than the others, while only the thigh meat of Pekin duck had the highest activity. Cooking inactivated CAT and decreased the activity of GPx and SOD. The thigh meat of Woorimatdak, White Leghorn, Yeonsan ogye and Hyunin black contained more total iron than the breast meat of those breeds. The heme-iron lost during cooking ranged from 3.2% to 14.8%. It is noted that the thigh meat had higher thiobarbituric acid reactive substances values than the breast in all chicken breeds. Though Woorimatdak showed higher antioxidant enzyme activity and lower released-iron percentage among Korean native chickens, no differences were found on lipid oxidation. We confirm that the dark meat of poultry exhibited higher antioxidant enzyme activity and contained more iron than the white meat. PMID:26954148

  7. Characterization and 2D NMR study of the stable [9-21, 15-27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II.

    PubMed Central

    Le-Nguyen, D.; Heitz, A.; Chiche, L.; el Hajji, M.; Castro, B.

    1993-01-01

    The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment. PMID:8443596

  8. Trypsin-enabled construction of anti-fouling and self-cleaning polyethersulfone membrane.

    PubMed

    Shi, Qing; Su, Yanlei; Ning, Xue; Chen, Wenjuan; Peng, Jinming; Jiang, Zhongyi

    2011-01-01

    Constructing anti-fouling and self-cleaning membrane surfaces based on covalent attachment of trypsin on poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES) membrane was reported. The carboxylic acid groups enriched on asymmetric PMAA-g-PES membrane surface were activated with 1-ethyl-(3-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) and employed as chemical anchors for the conjugation with amino groups of trypsin. Activity assays showed that such chemically immobilized trypsin was much more active and stable than that of the physically adsorbed counterpart. Trypsin covalently attached on membrane surface could substantially resist protein fouling in dynamic flow process. The considerable enhancement of protein solution permeation flux was observed as a consequence of rapid enzymatic degradation of protein deposited onto membrane surface. The permeation flux of the membrane could be recovered upon simple hydraulic flush after protein filtration, suggesting superior self-cleaning property. After multi-cycle BSA filtration over 15-day period, the active self-cleaning membrane maintained more than 95.0% of its initial flux.

  9. Isolation of a new trypsin inhibitor from the Faba bean (Vicia faba cv. Giza 843) with potential medicinal applications.

    PubMed

    Fang, Evandro Fei; Hassanien, Abdallah Abd Elazeem; Wong, Jack Ho; Bah, Clara Shui Fern; Soliman, Saeed Saad; Ng, Tzi Bun

    2011-01-01

    A new 15-kDa Bowman-Birk type trypsin inhibitor (termed VFTI-G1) was isolated from the seeds of Faba bean (Vicia faba cv. Giza 843) using cation exchange chromatography on an SP-Sepharose column, anion exchange chromatography on Q-Sepharose and Mono Q columns, and finally size exclusion chromatography on a Superdex 75 column. VFTI-G1 manifested significant antiproteolytic activity against trypsin (5761 BAEE units/mg, K(i) 20.4 × 10(-9) M), but only a slight chymotrypsin inhibitory activity (< 10 BTEE units/mg). The suitable environment to sustain its trypsin inhibitory activity was at temperatures below 60 °C and at pH 7. Its trypsin inhibitory activity was inhibited by the reducing agent dithiothreitol in a dose-dependent manner, indicating the significance of intact disulfide bonds to the trypsin inhibitory activity. It inhibited HIV-1 reverse transcriptase (RT) activity with an IC(50) of about 0.76 µM. Furthermore, VFTI-G1 showed specific antiproliferative activity toward HepG2 hepatoma cells by inducing chromatin condensation and cell apoptosis. The HIV-1 RT inhibitory activity of VFTI-G1 and its specific antiproliferative activity toward Hep G2 cells may find medical applications.

  10. Women's Class Strategies as Activism in Native Community Building in Toronto, 1950-1975

    ERIC Educational Resources Information Center

    Howard-Bobiwash, Heather

    2003-01-01

    Between the end of World War II and the early 1970s, many Native women in Ontario came to Toronto in the hopes of accessing higher education, jobs, and freedom denied them on reserves under the oppression of federal government tutelage. However, much of the literature on Native rural-urban migration in Canada concentrates on an association between…

  11. IL-1β is a key cytokine that induces trypsin upregulation in the influenza virus-cytokine-trypsin cycle.

    PubMed

    Indalao, I L; Sawabuchi, T; Takahashi, E; Kido, H

    2017-01-01

    Severe influenza is characterized by a cytokine storm, and the influenza virus-cytokine-trypsin cycle is one of the important mechanisms of viral multiplication and multiple organ failure. The aim of this study was to define the key cytokine(s) responsible for trypsin upregulation. Mice were infected with influenza virus strain A/Puerto Rico/8/34 (H1N1) or treated individually or with a combination of interleukin-1β, interleukin-6, and tumor necrosis factor α. The levels of these cytokines and trypsin in the lungs were monitored. The neutralizing effects of anti-IL-1β antibodies on cytokine and trypsin expression in human A549 cells and lung inflammation in the infected mice were examined. Infection induced interleukin-1β, interleukin-6, tumor necrosis factor α, and ectopic trypsin in mouse lungs in a dose- and time-dependent manner. Intraperitoneal administration of interleukin-1β combined with other cytokines tended to upregulate trypsin and cytokine expression in the lungs, but the combination without interleukin-1β did not induce trypsin. In contrast, incubation of A549 cells with interleukin-1β alone induced both cytokines and trypsin, and anti-interleukin-1β antibody treatment abrogated these effects. Administration of the antibody in the infected mice reduced lung inflammation area. These findings suggest that IL-1β plays a key role in trypsin upregulation and has a pathological role in multiple organ failure.

  12. A designed glycoprotein analogue of Gc-MAF exhibits native-like phagocytic activity.

    PubMed

    Bogani, Federica; McConnell, Elizabeth; Joshi, Lokesh; Chang, Yung; Ghirlanda, Giovanna

    2006-06-07

    Rational protein design has been successfully used to create mimics of natural proteins that retain native activity. In the present work, de novo protein engineering is explored to develop a mini-protein analogue of Gc-MAF, a glycoprotein involved in the immune system activation that has shown anticancer activity in mice. Gc-MAF is derived in vivo from vitamin D binding protein (VDBP) via enzymatic processing of its glycosaccharide to leave a single GalNAc residue located on an exposed loop. We used molecular modeling tools in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle (alpha3W, PDB entry 1LQ7). The resulting 69-residue model peptide, MM1, has been successfully synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) form. Circular dichroism spectroscopy confirmed the expected alpha-helical secondary structure. The thermodynamic stability as evaluated from chemical and thermal denaturation is comparable with that of the scaffold protein, alpha3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the macrophage stimulation activity of natural Gc-MAF; in vitro tests show an identical enhancement of Fc-receptor-mediated phagocytosis in primary macrophages. GalNAc-MM1 provides a framework for the development of mutants with increased activity that could be used in place of Gc-MAF as an immunomodulatory agent in therapy.

  13. Preparation of native cellulose-AgCl fiber with antimicrobial activity through one-step electrospinning.

    PubMed

    Wang, Shaojun; Zhang, Xiaomin; Luo, Ting; Zhu, Jin; Su, Shengpei

    2017-02-01

    The native Cellulose-AgCl fiber have been firstly fabricated by one-step electrospinning of cellulose solution with poly(vinyl pyrrolidone) (PVP) and AgNO3. X-ray diffraction, Scanning electron microscopy (SEM), Energy dispersive spectrometer, Thermo-gravimetric analysis and Fourier transform infrared are used to characterize the crystal structure, morphology and composition of cellulose-AgCl nanocomposites. The results of SEM indicate that the size of AgCl in cellulose fiber matrix is able to be adjusted by the addition of Polyvinylpyrrolidone (PVP). The antimicrobial activity of the nanocomposites fiber is also tested against the model microbes E. coli (Gram-negative) and S. aureus (Gram-positive). The results indicate that cellulose-AgCl nanocomposites have a good antimicrobial activity, which is improving with the decrease of AgCl size in fiber matrix. This work provides a novel and simple way to adjust the AgCl size in electrospinning cellulose matrix which can be applied as functional biomaterials.

  14. The investigation of antibacterial activity of selected native plants from North of Iran

    PubMed Central

    Koohsari, H; Ghaemi, EA; Sadegh Sheshpoli, M; Jahedi, M; Zahiri, M

    2015-01-01

    Plant derived products have been used for medicinal purposes during centuries. Bacterial resistance to currently used antibiotics has become a concern to public health. The development of bacterial super resistant strains has resulted in the currently used antibiotic agents failing to end many bacterial infections. For this reason, the search is ongoing for new antimicrobial agents, both by the design and by the synthesis of new agents, or through the search of natural sources for yet undiscovered antimicrobial agents. Herbal medications in particular have seen a revival of interest due to a perception that there is a lower incidence of adverse reactions to plant preparations compared to synthetic pharmaceuticals. Coupled with the reduced costs of plant preparations, this makes the search for natural therapeutics an attractive option. This research was carried out to assess the antibacterial activity aqueous and ethanolic extracts of six Azadshahr township Native plants in north of Iran against six species of pathogen bacteria by using three methods of Disk diffusion, Well method and MBC. The results of this research indicated that the effect of ethanol extracts were more than aqueous extract and among six plants, Lippia citriodora and Plantago major ethanol extract had the most antibacterial activity in any of the three methods. Gram-positive bacteria were more sensitive than gram-negative bacteria. Staphylococcus epidermidis and Staphylococcus aureus were the most susceptible Gram-positive bacteria.

  15. Functional constituents and antioxidant activities of eight Chinese native goji genotypes.

    PubMed

    Zhang, Qiuyun; Chen, Weiwei; Zhao, Jianhua; Xi, Wanpeng

    2016-06-01

    We quantified the levels of polyphenols, carotenoids and polysaccharides in fruits of the eight Chinese native goji genotypes, antioxidant activities of these fruit extracts were also evaluated by DPPH, ABTS and FRAP methods. Quercetin-rhamno-di-hexoside (435-1065 μg/g) and quercetin-3-O-rutinoside (159-629 μg/g) were found to be the predominant flavonoids. Chlorogenic acid was the most abundant phenolic acid (113-526 μg/g), while zeaxanthin (17-9306 μg/g) was the major carotenoid. The total antioxidant activities (TAA) of the berry extracts were significantly correlated with the total polysaccharide and phenolic contents, but not with total carotenoid (TC) levels. Overall, fruits of the Ningxia goji (Lycium barbarum L.) genotypes, DM (Damaye), NJ1 (Ningji No.1), BH (Baihua) and NH (Ningxiahuangguo) were not only rich in polyphenols, carotenoids and polysaccharides, but had significantly higher TAA than those of the other genotypes, suggesting that they represent an excellent source of antioxidants for human nutrition.

  16. Short communication: Assessing antihypertensive activity in native and model Queso Fresco cheeses.

    PubMed

    Paul, M; Van Hekken, D L

    2011-05-01

    Hispanic-style cheeses are one of the fastest growing varieties in the United States, making up approximately 2% of the total cheese production in this country. Queso Fresco is one of most popular Hispanic-style cheeses. Protein extracts from several varieties of Mexican Queso Fresco and model Queso Fresco were analyzed for potential antihypertensive activity. Many Quesos Frescos obtained from Mexico are made from raw milk and therefore the native microflora is included in the cheese-making process. Model Queso Fresco samples were made from pasteurized milk and did not utilize starter cultures. Water-soluble protein extracts from 6 Mexican Quesos Frescos and 12 model cheeses were obtained and assayed for their ability to inhibit angiotensin-converting enzyme, implying potential as foods that can help to lower blood pressure. All model cheeses displayed antihypertensive activity, but mainly after 8 wk of aging when they were no longer consumable, whereas the Mexican samples did display some angiotensin-converting enzyme inhibitory action after minimal aging.

  17. Walking Patterns in a Sample of African American, Native American, and Caucasian Women: The Cross-Cultural Activity Participation Study

    ERIC Educational Resources Information Center

    Whitt, Melicia C.; DuBose, Katrina D.; Ainsworth, Barbara E.; Tudor-Locke, Catrine

    2004-01-01

    This analysis describes walking patterns among African American, Native American, and Caucasian women from South Carolina and New Mexico. Walking was assessed using pedometer and physical activity (PA) record data based on 4 consecutive days on either three (Study Phase 1) or two (Study Phase 2) occasions. Participants walked 5,429 [plus or minus]…

  18. Activity patterns and parasitism rates of fire ant decapitating flies (Diptera:Phoridae:Pseudacteon spp.) in their native Argentina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: This work describes the annual and daily activity patterns of two parasitoid fly communities of the fire ant S. invicta (Hymenoptera: Formicidae) in their native Argentina. Pseudacteon (Diptera: Phoridae) flies were censused monthly for one year at two sites in northwestern Corr...

  19. Stages of Learning: Building a Native Curriculum. Teachers' Guide, Student Activities--Part I, Research Unit--Part II.

    ERIC Educational Resources Information Center

    Candline, Mary

    This language arts curriculum developed for Native American students in Manitoba (Canada) consists of a teachers' guide, a student guide, and a research unit. The curriculum includes reading selections and learning activities appropriate for the different reading levels of both upper elementary and secondary students. The purpose of the unit is…

  20. Are valve repairs associated with better outcomes than replacements in patients with native active valve endocarditis?

    PubMed

    Zhao, Dong; Zhang, Benqing

    2014-12-01

    A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was whether valve replacement was associated with higher morbidity and mortality rates than valve repair in patients with native active valve endocarditis. Altogether 662 papers were found using the reported search, of which 7 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. Traditionally, valve replacement has been the standard therapy for valve endocarditis when surgical treatment is indicated. But now valve repair is increasingly used as an alternative, which may avoid disadvantages of anticoagulation, lower the risk of prosthetic infection and improve postoperative survival. To compare outcomes of these two treatments between studies can be difficult because most of related papers contain raw data on prosthetic valve endocarditis or healed endocarditis, which were excluded from our manuscript. Studies only analysing the outcomes of either of these treatments without the comparison of valve repair and replacement were also excluded. Finally, seven papers were identified. The American Heart Association/American College of Cardiology 2006 valvular guidelines recommended that mitral valve repair should be performed instead of replacement when at all possible. In three of the seven studies, there were significant differences between valve repair and replacement in long-term survival. One study found that aortic valve repair offered better outcomes in freedom from reoperation at 5 years (P = 0.021) and in survival at 4 years (repair vs replacement 88 vs 65%; P = 0.047). One study reported that there was improved event-free survival at 10 years in the mitral valve repair group (P = 0.015), although there was more previous septic embolization in this group. In one study, early and late mortality

  1. Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata).

    PubMed

    Wilson, K A; Tan-Wilson, A L

    1987-05-01

    The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.

  2. Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity.

    PubMed

    Kim, S B; Ki, K S; Khan, M A; Lee, W S; Lee, H J; Ahn, B S; Kim, H S

    2007-09-01

    This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100 degrees C) WPC (2% protein solution) were incubated at 50 degrees C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The alpha-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and alpha-lactalbumin. The beta-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The beta-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of beta-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that

  3. Study on the interaction of β-carotene and astaxanthin with trypsin and pepsin by spectroscopic techniques.

    PubMed

    Li, Xiangrong; Li, Peihong

    2016-05-01

    β-Carotene and astaxanthin are two carotenoids with powerful antioxidant properties, but the binding mechanisms of β-carotene/astaxanthin to proteases remain unclear. In this study, the interaction of these two carotenoids with trypsin and pepsin was investigated using steady-state and time-resolved fluorescence measurements, synchronous fluorescence spectroscopy, UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The experimental results indicated that the quenching mechanisms of trypsin/pepsin by the two carotenoids are static processes. The binding constants of trypsin and pepsin with these two carotenoids are in the following order: astaxanthin-trypsin > astaxanthin-pepsin > β-carotene-trypsin > β-carotene-pepsin, respectively. Thermodynamic investigations revealed that the interaction between the two carotenoids and trypsin/pepsin is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. In addition, as shown by synchronous fluorescence spectroscopy, UV-vis absorption spectroscopy and CD, the two carotenoids may induce conformational and microenvironmental changes in trypsin/pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of the two carotenoids with trypsin/pepsin and is helpful in understanding their effect on protein function and their biological activity in vivo.

  4. Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

    PubMed

    Moulin, M M; Rodrigues, R; Ribeiro, S F F; Gonçalves, L S A; Bento, C S; Sudré, C P; Vasconcelos, I M; Gomes, V M

    2014-11-07

    Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

  5. Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates.

    PubMed Central

    Ohta, K; Makinen, K K; Loesche, W J

    1986-01-01

    An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibly inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethylchloromethyl ketone, alpha-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-alpha-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine. Images PMID:3013780

  6. Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods.

    PubMed

    Li, Xiangrong; Ni, Tianjun

    2016-06-01

    α-Tocopherol is a required nutrient for a variety of biological functions. In this study, the binding of α-tocopherol to trypsin and pepsin was investigated using isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements, circular dichroism (CD) spectroscopy, and molecular modeling methods. Thermodynamic investigations reveal that α-tocopherol binds to trypsin/pepsin is synergistically driven by enthalpy and entropy. The fluorescence experimental results indicate that α-tocopherol can quench the fluorescence of trypsin/pepsin through a static quenching mechanism. The binding ability of α-tocopherol with trypsin/pepsin is in the intermediate range, and one molecule of α-tocopherol combines with one molecule of trypsin/pepsin. As shown by circular dichroism (CD) spectroscopy, α-tocopherol may induce conformational changes of trypsin/pepsin. Molecular modeling displays the specific binding site and gives information about binding forces and α-tocopherol-tryptophan (Trp)/tyrosine (Tyr) distances. In addition, the inhibition rate of α-tocopherol on trypsin and pepsin was studied. The study provides a basic data set for clarifying the binding mechanisms of α-tocopherol with trypsin and pepsin and is helpful for understanding its biological activity in vivo.

  7. Anticoagulant activity of native and partially degraded glycoglucuronomannan after chemical sulfation.

    PubMed

    de Oliveira Barddal, Helyn Priscila; Gracher, Ana Helena Pereira; Simas-Tosin, Fernanda Fogagnoli; Iacomini, Marcello; Cipriani, Thales Ricardo

    2015-09-01

    Heparin has great clinical importance as anticoagulant and antithrombotic agent. However, because of its risks of causing bleeding and contamination by animal pathogens, several studies aim to obtain alternatives to heparin. In the search for anticoagulant and antithrombotic agents from a non-animal source, a glycoglucuronomannan from the gum exudate of the plant Vochysia thyrsoidea was partially hydrolyzed, and both native and partially degraded polysaccharides were chemically sulfated, yielding VThS and Ph-VThS respectively. Methylation analysis indicated that sulfation occurred preferentially at the O-5 position of arabinose units in the VThS and at the O-6 position of mannose units in Ph-VThS. In vitro aPTT assay showed that VThS and Ph-VThS have anticoagulant activity, which could be controlled by protamine, and ex vivo aPTT assay demonstrated that Ph-VThS is absorbed by subcutaneous route. Like heparin, they were able to inhibit α-thrombin and factor Xa by a serpin-dependent mechanism. In vivo, VThS and Ph-VThS reduced thrombus formation by approximately 50% at a dose of 40 IU/kg, similarly to heparin. The results demonstrated that the chemically sulfated polysaccharides are promising anticoagulant and antithrombotic agents.

  8. Anti-tumoral effects of a trypsin inhibitor derived from buckwheat in vitro and in vivo.

    PubMed

    Bai, Chong-Zhi; Feng, Ma-Li; Hao, Xu-Liang; Zhao, Zhi-Juan; Li, Yu-Ying; Wang, Zhuan-Hua

    2015-08-01

    Native buckwheat, a common component of food products and medicine, has been observed to inhibit cancer cell proliferation in vitro. The aim of the present study was to evaluate the in vitro and in vivo anti-tumoral effects of recombinant buckwheat trypsin inhibitor (rBTI) on hepatic cancer cells and the mechanism of apoptosis involved. Apoptosis in the H22 cell line induced by rBTI was identified using MTT assays, DNA electrophoresis, flow cytometry, morphological observation of the nuclei, measurement of cytochrome C and assessment of caspase activation. It was identified that rBTI decreases cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. rBTI-induced apoptosis occurred in association with mitochondrial dysfunction, leading to the release of cytochrome C from the mitochondria to the cytosol, as well as the activation of caspase-3, -8 and -9. In conclusion, the results of the present study suggested that rBTI specifically inhibited the growth of the H22 hepatic carcinoma cell line in vitro and in vivo in a concentration-dependent and time-dependent manner, while there were minimal effects on the 7702 normal liver cell line. In addition, rBTI‑induced apoptosis in H22 cells was, at least in part, mediated by a mitochondrial pathway via caspase-9.

  9. Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.

    PubMed

    Xu, Fei; Wang, Wei-Han; Tan, Yu-Jing; Bruening, Merlin L

    2010-12-15

    Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

  10. Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site.

    PubMed

    Pidugu, Lakshmi Swarna Mukhi; Neu, Heather; Wong, Tin Lok; Pozharski, Edwin; Molloy, John L; Michel, Sarah L J; Toth, Eric A

    2017-04-01

    3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2 to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally and via inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.

  11. Native Knowledge in the Americas.

    ERIC Educational Resources Information Center

    Kidwell, Clara Sue

    1985-01-01

    Native American science is defined as activities of native peoples of the New World in observing physical phenomena and attempting to explain and control them. Problems in studying native science, ethnoscience and native science, archaeostronomy and ethnoastronomy, ethnobotany, agriculture, technology, and future directions are discussed. (JN)

  12. The diamondback moth, Plutella xylostella, specifically inactivates Mustard Trypsin Inhibitor 2 (MTI2) to overcome host plant defence.

    PubMed

    Yang, Limei; Fang, Zhiyuan; Dicke, Marcel; van Loon, Joop J A; Jongsma, Maarten A

    2009-01-01

    The mustard trypsin inhibitor family has so far only been described among cruciferous species which represent the host plants for the specialist diamondback moth (DBM), Plutella xylostella. The performance of a Dutch and Chinese strain of DBM was assessed on transgenic Arabidopsis expressing Mustard Trypsin Inhibitor 2 (MTI2) at a level of 84 microg/g fresh weight equivalent to 12 microM. No significant differences in larval mortality or development were found relative to the control. Trypsin activity in gut extracts from larvae feeding on either control or transgenic plants were titrated with MTI2 and SKTI (Soybean Kunitz Trypsin Inhibitor) to assess the basis of the insensitivity to MTI2. The specific trypsin activity per gut of larvae reared on MTI2 plants was not significantly higher compared to the control, and ca. 80% of trypsin activity could be inhibited by both inhibitors in both treatments, suggesting no specific induction of PI-insensitive activity in response to MTI2 in the diet. On the basis of the apparent equilibrium dissociation constant of Plutella trypsins for MTI2 (80 nM), the gut trypsin concentration (4.8 microM), and the MTI2 concentration in the leaves (12 microM) it was calculated that 99% of the gut trypsin activity sensitive to MTI2 should be inhibited in vivo, unless MTI2 was degraded. Indeed, we found that a pre-incubation of MTI2 and SKTI with gut proteases for 3 h resulted in complete loss of inhibitory activity of MTI2, but not of SKTI, at the concentration ratios found in planta. This process was enzymatic as it was inactivated by heat. Gut extracts of larvae reared on control or MTI2 leaves were equally well capable of this degradation indicating that the inactivating enzymes are constitutively expressed. In conclusion, it appears that the insensitivity of the diamondback moth to MTI2 can be sufficiently explained by the specific degradation of MTI2, thereby protecting itself against this protease inhibitor which is part of the

  13. Levels of toxic arsenic species in native terrestrial plants from soils polluted by former mining activities.

    PubMed

    García-Salgado, Sara; Quijano, M Ángeles

    2014-03-01

    Ten native terrestrial plants from soils polluted by former mining activities (Mónica mine, NW Madrid, Spain), with high total arsenic concentration levels (up to 3500 μg g(-1)), have been studied to determine the fraction of arsenic present as toxic forms (inorganic and methylated species), which present a higher mobility and therefore the potential risk associated with their reintegration into the environment is high. Roots and aboveground parts were analyzed separately to assess possible transformations from translocation processes. Extractions were carried out with deionized water by microwave-assisted extraction at a temperature of 90 °C and three extraction steps of 7.5 min each. Total extracted arsenic concentrations were determined by inductively coupled plasma atomic emission spectrometry, showing extraction percentages from 9 to 39% (calculated as the ratio between total extracted arsenic (Asext) and total arsenic (AsT) concentrations in plants). Speciation studies, performed by high performance liquid chromatography-photo-oxidation-hydride generation-atomic fluorescence spectrometry, showed the main presence of arsenate (As(v)) (up to 350 μg g(-1)), followed by arsenite (As(iii)), in both plant parts. Monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO) were also found only in some plants. On the other hand, the use of 0.5 mol L(-1) acetic acid as an extractant led to higher extraction percentages (33-87%), but lower column recoveries, probably due to the extraction of arsenic compounds different to the toxic free ions studied, which may come from biotransformation mechanisms carried out by plants to reduce arsenic toxicity. However, As(v) concentrations increased up to 800 μg g(-1) in acid medium, indicating the probable release of As(v) from organoarsenic compounds and therefore a higher potential risk for the environment.

  14. Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

    PubMed

    Pereira, Keitty R B; Botelho-Júnior, Sylvio; Domingues, Dalvania P; Machado, Olga L T; Oliveira, Antônia E A; Fernandes, Kátia V S; Madureira, Herika C; Pereira, Telma N S; Jacinto, Tânia

    2011-11-01

    In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.

  15. TRADITIONAL FOODS AND PHYSICAL ACTIVITY PATTERNS AND ASSOCIATIONS WITH CULTURAL FACTORS IN A DIVERSE ALASKA NATIVE POPULATION

    PubMed Central

    Redwood, Diana G; Ferucci, Elizabeth D; Schumacher, Mary C; Johnson, Jennifer S; Lanier, Anne P; Helzer, Laurie J; Tom-Orme, Lillian; Murtaugh, Maureen A; Slattery, Martha L

    2010-01-01

    Objectives To determine the prevalence of traditional food and physical activity use and associations with cultural factors among 3,830 Alaska Native and American Indian (AN/AI) people enrolled in the Education and Research Towards Health (EARTH) Study in 3 regions of Alaska. Study design Cross-sectional analysis of baseline data from a cohort study. Methods Participants (2,323 women and 1,507 men) completed a computer-assisted self-administered questionnaire that included information on diet, physical activity, life-style and cultural factors. Results Over 92% of participants reported eating at least 1 traditional food in the past year. The top 3 traditional foods reported were fish, moose and agutaq (a mixture of berries and fat). The percentage of people who consumed traditional foods varied by region and age but not by sex (p<0.01). Almost 70% of participants engaged in at least one traditional harvesting physical activity. Picking berries or greens, cutting/smoking fish or meat and fishing were the most common activities. Participation in traditional physical activity was highest in south-west Alaska and was higher among men than women, but did not differ by age (p<0.01). Both traditional food and physical activity were associated with greater tribal self-identification, speaking a Native language at home, using traditional remedies and participating in or attending traditional events (p<0.05). Conclusions The EARTH Study found relationships between traditional food use, physical activities, cultural activities and behaviours. Consumption of a variety of traditional foods and participation in traditional physical activities remain an important part of the contemporary Alaska Native life-style. Efforts to promote and sustain these foods and activities in AN/AI populations may lead to improved health outcomes. PMID:19024803

  16. Use of denatured radioalbumin for determination of trypsin and chymotrypsin inhibitors in different plant seeds

    SciTech Connect

    Blahovec, J. )

    1991-02-01

    The procedure for determination of trypsin and chymotrypsin inhibitors with urea-denatured albumin labeled by {sup 125}I is described. The content of both types of inhibitory activity has been determined in crude extracts of soybean, bean, lentil, pea, horse bean, maize, and 20 pea cultivars. The method is sufficiently sensitive, reliable, and particularly suitable when estimations must be done in crude plant extract with low inhibitory activity.

  17. Could Early Surgery Get Beneficial in Adult Patients with Active Native Infective Endocarditis? A Meta-Analysis

    PubMed Central

    Jia, Liqun; Wang, Zanxin; Fu, Qiang

    2017-01-01

    After a thorough search through the database as PubMed database and Embase database, the clinical experimental articles have been selected out on the effects of early surgery on the treatment of active native infective endocarditis. The quality of the trials included in this study was assessed by researcher according to the Cochrane Handbook for Systematic Reviews of Interventions, version 5.1.0. A meta-analysis was carried out in terms of clinical efficacy criteria by RevMan 5.3 software. Based on the results, we cautiously conclude that early surgery used for active native infective endocarditis could reduce in-hospital mortality, follow-up mortality, and IE-related mortality. PMID:28326318

  18. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    SciTech Connect

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-08-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.

  19. Development of an open-tubular trypsin reactor for on-line digestion of proteins

    PubMed Central

    de Jong, G. J.; van Bennekom, W. P.

    2007-01-01

    A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm−2 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL−1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening. PMID:17899035

  20. Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

    NASA Technical Reports Server (NTRS)

    Kristjansson, H.; Hochstein, L. I.

    1986-01-01

    Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

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

    PubMed

    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.

  2. Screening of Caatinga plants as sources of lectins and trypsin inhibitors.

    PubMed

    Arcoverde, José Hélton Vasconcelos; Carvalho, Aline de Souza; Neves, Fernanda Pacífico de Almeida; Dionízio, Bianca Paiva; Pontual, Emmanuel Viana; Paiva, Patrícia Maria Guedes; Napoleão, Thiago Henrique; Correia, Maria Tereza dos Santos; da Silva, Márcia Vanusa; Carneiro-da-Cunha, Maria das Graças

    2014-01-01

    Although it is one of the most threatened areas in the Earth, there are few studies on the biotechnological potential of the Caatinga. This work evaluated 36 extracts from 27 Caatinga plants for lectin and trypsin inhibitor activities. The presence of lectin was detected in 77.7% of samples by haemagglutinating assay. The highest values of specific haemagglutinating activity were found in extracts of leaves from Mimosa lewesii, Bauhinia acuruana and Manilkara rufula and in branches from Myracrodruon urundeuva. Trypsin inhibitor activity was detected in 63.9% of the tested extracts, strong inhibitory effect (>70%) being found in 11 samples. This work demonstrates that Caatinga is a potential source of bioactive plant proteins that can be isolated and studied for several applications. The biochemical prospecting of Caatinga is essential for collection of bioactive principles so as to add conservation value to the region.

  3. The effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral IgG1.

    PubMed Central

    Brock, J H; Arzabe, F R; Ortega, F; Piñeiro, A

    1977-01-01

    Limited proteolysis of bovine colostral IgG1 by trypsin caused loss of specific antibody activity but column chromatography showed that relatively little cleavage into fragements had occurred. Polyacrlamide-agarose SDS electrophoresis of the 2-mercaptoethanol-treated digest revealed, however, that extensive cleavage of light chains had occurred even though most of the material before reduction had a mol. wt close to that of undigested IgG1. Although a Fab-type fragment was detected in the digest by immunoelectrophoresis it appeared to be only a minor component. Chymotrypsin had little effect upon either the structure or antibody activity of IgG1. These findings may explain the effect of trypsin and chymotrypsin on the bactericidal activity of colostral antibodies. Images Figure 2 Figure 3 PMID:321343

  4. Impact of Native and Invasive Earthworm Activity on Forest Soil Organic Matter Dynamics

    NASA Astrophysics Data System (ADS)

    Top, Sara; Filley, Timothy

    2010-05-01

    Many northern North American forests are experiencing the introduction of exotic European lumbricid species earthworms with documented losses in litter layers, expansion of A-horizons, loss of the organic horizon, changes in fine root density, and shifts in microbial populations as a result. Some of these forests were previously devoid of these ecosystem engineers. We compare the soil isotope and molecular chemistry from two free air CO2 enrichment (FACE) forest experiments (aspen FACE at Rhinelander, Wisconsin and sweet gum FACE at Oak Ridge National Lab, Tennessee) that lie within the zones of earthworm invasion. These sites exhibit differences in amounts of exotic and native species as well as endogeic (predominantly mineral soil dwelling) and epigeic (litter and organic matter horizon dwelling) types. We investigated the impact of earthworm activity by tracking the relative abundance and stable carbon isotope compositions of lignin and substituted fatty acids extracted from isolated earthworms and their fecal pellets and from host soils. Additionally, 15N-labeled additions to the soil provide additional methods for tracking earthworm impacts. Indications of root vs leaf input to earthworm casts and fecal matter were derived from differences in the chemical composition of cutin, suberin, and lignin. The isotopically depleted CO2 used in FACE and the resulting isotopically depleted plant organic matter afford an excellent opportunity to assess biopolymer-specific turnover dynamics. We find that endogeic species are proportionately more responsible for fine root cycling while some epigeic species are responsible for microaggregation of foliar cutin. CSIA of fecal pellet lignin and SFA indicate how these biopolymer pools can be derived from variable sources, roots, background soil, foliar tissue within one earthworm. Additionally, CSIA indicates the distinct roles that different earthworm types have in "aging" surface soil biopolymer pools through encapsulation and

  5. Increased potential for wound activated production of Prostaglandin E2 and related toxic compounds in non-native populations of Gracilaria vermiculophylla.

    PubMed

    Hammann, Mareike; Rempt, Martin; Pohnert, Georg; Wang, Gaoge; Boo, Sung Min; Weinberger, Florian

    2016-01-01

    The capacity of the East Asian seaweed Gracilaria vermiculophylla ("Ogonori") for production of prostaglandin E2 from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE2, in 90% more PGE2, in 37% more PGA2 and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE2, but also PGA2 and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.

  6. Genetic diversity and antifungal activity of native Pseudomonas isolated from maize plants grown in a central region of Argentina.

    PubMed

    Cordero, Paula; Cavigliasso, Andrea; Príncipe, Analía; Godino, Agustina; Jofré, Edgardo; Mori, Gladys; Fischer, Sonia

    2012-07-01

    Pseudomonas strains producing antimicrobial secondary metabolites play an important role in the biocontrol of phytopathogenic fungi. In this study, native Pseudomonas spp. isolates were obtained from the rhizosphere, endorhizosphere and bulk soil of maize fields in Córdoba (Argentina) during both the vegetative and reproductive stages of plant growth. However, the diversity based on repetitive-element PCR (rep-PCR) and amplified ribosomal DNA restriction analysis (ARDRA) fingerprinting was not associated with the stage of plant growth. Moreover, the antagonistic activity of the native isolates against phytopathogenic fungi was evaluated in vitro. Several strains inhibited members of the genera Fusarium, Sclerotinia or Sclerotium and this antagonism was related to their ability to produce secondary metabolites. A phylogenetic analysis based on rpoB or 16S rRNA gene sequences confirmed that the isolates DGR22, MGR4 and MGR39 with high biocontrol potential belonged to the genus Pseudomonas. Some native strains of Pseudomonas were also able to synthesise indole acetic acid and to solubilise phosphate, thus possessing potential plant growth-promoting (PGPR) traits, in addition to their antifungal activity. It was possible to establish a relationship between PGPR or biocontrol activity and the phylogeny of the strains. The study allowed the creation of a local collection of indigenous Pseudomonas which could be applied in agriculture to minimise the utilisation of chemical pesticides and fertilisers.

  7. Explicit and implicit second language training differentially affect the achievement of native-like brain activation patterns.

    PubMed

    Morgan-Short, Kara; Steinhauer, Karsten; Sanz, Cristina; Ullman, Michael T

    2012-04-01

    It is widely believed that adults cannot learn a foreign language in the same way that children learn a first language. However, recent evidence suggests that adult learners of a foreign language can come to rely on native-like language brain mechanisms. Here, we show that the type of language training crucially impacts this outcome. We used an artificial language paradigm to examine longitudinally whether explicit training (that approximates traditional grammar-focused classroom settings) and implicit training (that approximates immersion settings) differentially affect neural (electrophysiological) and behavioral (performance) measures of syntactic processing. Results showed that performance of explicitly and implicitly trained groups did not differ at either low or high proficiency. In contrast, electrophysiological (ERP) measures revealed striking differences between the groups' neural activity at both proficiency levels in response to syntactic violations. Implicit training yielded an N400 at low proficiency, whereas at high proficiency, it elicited a pattern typical of native speakers: an anterior negativity followed by a P600 accompanied by a late anterior negativity. Explicit training, by contrast, yielded no significant effects at low proficiency and only an anterior positivity followed by a P600 at high proficiency. Although the P600 is reminiscent of native-like processing, this response pattern as a whole is not. Thus, only implicit training led to an electrophysiological signature typical of native speakers. Overall, the results suggest that adult foreign language learners can come to rely on native-like language brain mechanisms, but that the conditions under which the language is learned may be crucial in attaining this goal.

  8. Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

    PubMed Central

    Silva, José A.; Pompeu, Dávia G.; Smolka, Marcus B.; Gozzo, Fabio C.; Comar, Moacyr; Eberlin, Marcos N.; Marangoni, Sérgio

    2015-01-01

    In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge. PMID:26207098

  9. The Relationship between Active Hand and Ear Advantage in the Native and Foreign Language

    ERIC Educational Resources Information Center

    Mildner, V.; Stankovic, D.; Petkovic, M.

    2005-01-01

    In an experimental design involving two auditorily presented competing commands (one to each ear), 144 right-handed subjects (72 male and 72 female) were asked to provide motor responses. Half of each group of subjects was responding with their right hand and the other half with the left. The test was applied in the subjects' native language…

  10. Mediated Vocabulary in Native Speaker-Learner Interactions during an Oral Portfolio Activity

    ERIC Educational Resources Information Center

    Tocaimaza-Hatch, C. Cecilia

    2016-01-01

    This project investigated vocabulary learning from a sociocultural perspective--in particular, the way in which lexical knowledge was mediated in Spanish second language (L2) learners' and native speakers' (NSs') interactions. Nine students who were enrolled in an advanced conversation course completed an oral portfolio assignment consisting of…

  11. Project NAME: Native American Mathematics Education. Recommended Lesson Activities with Authentic Assessments.

    ERIC Educational Resources Information Center

    2001

    A project sought to improve mathematics instruction at the Winnebago Public School (WPS) on the Winnebago Indian Reservation (Nebraska) and to provide purposeful interactions between preservice teachers from Wayne State College and Native American children. WPS educators, grades K-6, improved their mathematics instructional ability by attending…

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

    PubMed

    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.

  13. Free radical scavenging and tyrosinase inhibition activity of oils and sericin extracted from Thai native silkworms (Bombyx mori).

    PubMed

    Manosroi, Aranya; Boonpisuttinant, Korawinwich; Winitchai, Supanida; Manosroi, Worapaka; Manosroi, Jiradej

    2010-08-01

    Oils and sericin were extracted from pupae and silk cocoons, respectively, of the five Thai native silkworms (Bombyx mori, Linnaeus (Bombycidae)), namely, Keawsakol, Nangnoi, Somrong, Nangleung, and Noneruesee, which are variations of the same species. The oils were extracted by a hot process using Soxhlet apparatus and a cold process using petroleum ether, while sericin was extracted by basic hydrolysis and autoclaving. Sericin from the five Thai native silkworms showed free radical scavenging activity lower than the standard antioxidants (vitamin C, vitamin E, and BHT) by about 20-100-fold, but all oils gave higher activity than that of the standard linoleic acid by 11-22-fold. Oil extracted from Noneruesee by the cold process gave the highest DPPH scavenging activity, compared with other oil samples. All sericin samples showed tyrosinase inhibition activity with IC(50) values in the range of 1.2-18.76 mg/mL, but only oils from Noneruesee extracted by the hot process, and Nangleung, Somrong, and Noneruesee extracted by the cold process, showed this activity. Oil extracted by the hot process and sericin by basic hydrolysis from Noneruesee gave the highest tyrosinase inhibition activity, but lower than that of the standards vitamin C and kojic acid by 20-49 and 3-8 times, respectively. This study has suggested that sericin and oil from Noneruesee extracted by basic hydrolysis and the cold process, which gave the highest tyrosinase inhibition and free radical scavenging activity, respectively, can be applied in antiaging and whitening cosmetic products.

  14. Electronic structures of Ascaris trypsin inhibitor in solution

    NASA Astrophysics Data System (ADS)

    Zheng, Haoping

    2003-11-01

    The electronic structures of Ascaris trypsin inhibitor in solution are obtained by the first-principles, all-electron, ab initio calculation using the self-consistent cluster-embedding (SCCE) method. The inhibitor, made up of 62 amino acid residues with 912 atoms, has two three-dimensional solution structures: 1ata and 1atb. The calculated ground-state energy of structure 1atb is lower than that of structure 1ata by 6.12 eV. The active sites are determined and explained: only structure 1atb has a N terminal at residue ARG+31. This shows that the structure 1atb is the stable and active form of the inhibitor, which is in agreement with the experimental results. The calculation reveals that some parts of the inhibitor can be easily changed while the inhibitor’s biological activity may be kept. This kind of information may be helpful in fighting viruses such as AIDS, SARS, and flu, since these viruses have higher variability. The calculation offers an independent theoretical estimate of the precision of structure determination.

  15. Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

    SciTech Connect

    Page, Michael J.; Di Cera, Enrico

    2010-06-14

    Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional protein domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.

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

    PubMed

    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.

  17. Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry.

    PubMed

    Maximova, Ksenia; Trylska, Joanna

    2015-10-01

    Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis-Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37°C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis-Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.

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

    SciTech Connect

    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{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.

  19. Taste-active compound levels in Korean native chicken meat: The effects of bird age and the cooking process.

    PubMed

    Jayasena, Dinesh D; Jung, Samooel; Kim, Hyun Joo; Yong, Hae In; Nam, Ki Chang; Jo, Cheorun

    2015-08-01

    The effects of bird age and the cooking process on the levels of several taste-active compounds, including inosine 5'-monophosphate (IMP), glutamic acid, cysteine, reducing sugars, as well as oleic, linoleic, arachidonic, and docosahexaenoic acids (DHA), in the breast and leg meats from a certified meat-type commercial Korean native chicken (KNC) strain (Woorimatdag) were investigated. KNC cocks were raised under similar standard conditions at a commercial chicken farm, and breast and leg meats from birds of various ages (10, 11, 12, 13, and 14 wk; 10 birds/age group) were obtained. After raw and cooked meat samples were prepared, they were analyzed for the aforementioned taste-active compounds. Compared to the leg meat, KNC breast meat had higher levels of IMP, arachidonic acid, and DHA, but lower levels of the other taste-active compounds (P < 0.05). KNC meat lost significant amounts of all the taste-active compounds, excluding oleic and linoleic acids, during the cooking process (P < 0.05). However, bird age only had a minor effect on the levels of these taste-active compounds. The results of this study provide useful information regarding the levels of taste-active compounds in KNC meat from birds of different ages, and their fate during the cooking process. This information could be useful for selection and breeding programs, and for popularizing native chicken meat.

  20. Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants.

    PubMed

    Gokey, Trevor; Baird, Teaster T; Guliaev, Anton B

    2012-11-01

    Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.

  1. Trypsin-like enzyme from eggs of the ascidian (protochordate), Halocynthia roretzi. Purification, properties, and physiological role.

    PubMed

    Sawada, H; Kawahigashi, M; Yokosawa, H; Ishii, S

    1985-12-15

    A trypsin-like enzyme has been purified to apparent homogeneity from eggs of the ascidian, Halocynthia roretzi, by a procedure including column chromatography on diethylaminoethyl-cellulose, phenyl-Sepharose, and soybean trypsin inhibitor-immobilized Sepharose 4B. The molecular weight of the enzyme was estimated to be 31,000 and 33,000 by gel electrophoresis in sodium dodecyl sulfate under the reducing and the nonreducing conditions, respectively. The isoelectric point of the enzyme was 4.8. The pH optimum of the activity was 8.4. The enzyme was stable between pH 6 and 9 in the presence of 0.005% Brij 35 as a stabilizer. Substrate specificity of the purified enzyme was broad toward various peptidyl-arginine (or -lysine) 4-methylcoumaryl-7-amides and was similar to that of a trypsin-like enzyme found in the fertilization product. The purified enzyme was inhibited by diisopropyl fluorophosphate and a variety of trypsin inhibitors including leupeptin, but not, or scarcely, inhibited by p-chloromercuribenzoic acid, pepstatin, chymostatin, bestatin, elastatinal, and tosyl-phenylalanyl-chloromethane. The rankings in the potencies of leupeptin and its six analogs as the inhibitors of the purified enzyme were well correlated with those found in their inhibitory effects on the expansion of perivitelline space. Thus, the trypsin-like enzyme possibly present in the fertilization product participates in the expansion of perivitelline space of the egg during fertilization of the ascidian.

  2. The properties of covalently immobilized trypsin on soap-free P(MMA-EA-AA) latex particles.

    PubMed

    Kang, Kai; Kan, Chengyou; Yeung, Anthony; Liu, Deshan

    2005-04-19

    The covalent immobilization of trypsin onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55 degrees C and 8.5, both of which were higher than that of the free form. It was found that K(m) (Michaelis constant) was 45.7 mg . ml(-1) and V(max) (maximal reaction rate) was 793.0 microg . min(-1) for immobilized trypsin, compared to a K(m) of 30.0 mg . ml(-1) and a V(max) of 5 467.5 microg . min(-1) for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times.

  3. U.S. Geological Survey activities related to American Indians and Alaska Natives: Fiscal year 2006

    USGS Publications Warehouse

    Marcus, Susan M.

    2008-01-01

    In the late 1800s, John Wesley Powell, the second director of the U.S. Geological Survey (USGS), followed his interest in the tribes of the Great Basin and Colorado Plateau and studied their cultures, languages, and surroundings. From that early time, the USGS has recognized the importance of Native knowledge and living in harmony with nature as complements to the USGS mission to better understand the Earth. Combining traditional ecological knowledge with empirical studies allows the USGS and Native American governments, organizations, and people to increase their mutual understanding and respect for this land. The USGS is the earth and natural science bureau within the U.S. Department of the Interior (DOI). The USGS does not have regulatory or land management responsibilities.

  4. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.

    PubMed

    de Souza, Adriana F; Torquato, Ricardo J S; Tanaka, Aparecida S; Sampaio, Claudio A M

    2005-11-01

    A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.

  5. Novel glutaredoxin activity of the yeast prion protein Ure2 reveals a native-like dimer within fibrils.

    PubMed

    Zhang, Zai-Rong; Perrett, Sarah

    2009-05-22

    Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.

  6. Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

    PubMed Central

    2013-01-01

    Background Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish. Results A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K m , k cat, and k cat /K m values using BApNA as substrate were 0.689 mM, 6.9 s-1, and 10 s-1 mM-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions. Conclusions Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications. PMID:24112762

  7. Immobilization of trypsin on graphene oxide for microwave-assisted on-plate proteolysis combined with MALDI-MS analysis.

    PubMed

    Xu, Guobin; Chen, Xiaoyi; Hu, Jianhua; Yang, Pengyuan; Yang, Dong; Wei, Liming

    2012-06-21

    With an ultra-high surface area and abundant functional groups, graphene oxide (GO) provides an ideal substrate for the immobilization of trypsin. We demonstrated that trypsin could be immobilized on GO sheets assisted by polymers as molecular spacers to maintain the activity of the enzyme. And with the trypsin-linked GO as the enzyme immobilization probe, a novel microwave-assisted on-plate digestion method has been developed with subsequent analysis by MALDI-MS. The feasibility and performance of the digestion approach were demonstrated by the proteolysis of standard proteins. The results show that this novel approach substantially accelerated proteolysis and reduced the time required for traditional procedures involving on-plate enzymatic digestion and sample preparation prior to MALDI-MS analysis. The novel digestion approach is simple and efficient, offering great promise for high throughput protein identification.

  8. The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

    PubMed

    Bayineni, Venkata Krishna; Venkatesh, Krishna; Sahu, Chandan Kumar; Kadeppagari, Ravi-Kumar

    2016-04-01

    Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications.

  9. Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

    PubMed Central

    Ganesh, V.; Lee, A. Y.; Clardy, J.; Tulinsky, A.

    1996-01-01

    Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174

  10. Preparation and characterization of a packed bead immobilized trypsin reactor integrated into a PDMS microfluidic chip for rapid protein digestion.

    PubMed

    Kecskemeti, Adam; Gaspar, Attila

    2017-05-01

    This paper demonstrates the design, efficiency and applicability of a simple, inexpensive and high sample throughput microchip immobilized enzymatic reactor (IMER) for rapid protein digestion. The IMER contains conventional silica particles with covalently immobilized trypsin packed inside of a poly(dimethylsiloxane) (PDMS) microchip channel (10mm×1mm×35µm). The microchip consists of 9 different channels, enabling 9 simultaneous protein digestions. Trypsin was covalently immobilized using carbodiimide activation, the ideal trypsin/silica particle ratio (i.e. measured mass ratio before the immobilization reaction) was determined. The amount of immobilized trypsin was 10-15μg trypsin for 1mg silica particle. Migration times of CZE peptide maps showed good repeatability and reproducibility (RSD%=0.02-0.31%). The IMER maintained its activity for 2 months, in this period it was used effectively for rapid proteolysis. Four proteins (myoglobin, lysozyme, hemoglobin and albumin) in a wide size range (15-70kDa) were digested to demonstrate the applicability of the reactor. Their CZE peptide maps were compared to peptide maps obtained from standard in-solution digestion of the four proteins. The number of peptide peaks correlated well with the theoretically expected peptide number in both cases, the peak patterns of the electropherograms were similar, however, digestion with the microchip IMER requires only <10s, while in-solution digestion takes 16h. LC-MS/MS peptide mapping was also carried out, the four proteins were identified with satisfying sequence coverages (29-50%), trypsin autolysis peptides were not detected. The protein content of human serum was digested with the IMER and with in-solution digestion.

  11. Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin.

    PubMed

    Mozhaev, V V; Siksnis, V A; Melik-Nubarov, N S; Galkantaite, N Z; Denis, G J; Butkus, E P; Zaslavsky BYu; Mestechkina, N M; Martinek, K

    1988-04-05

    This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.

  12. A Rapid Screening Analysis of Antioxidant Compounds in Native Australian Food Plants Using Multiplexed Detection with Active Flow Technology Columns.

    PubMed

    Rupesinghe, Emmanuel Janaka Rochana; Jones, Andrew; Shalliker, Ross Andrew; Pravadali-Cekic, Sercan

    2016-01-20

    Conventional techniques for identifying antioxidant and phenolic compounds in native Australian food plants are laborious and time-consuming. Here, we present a multiplexed detection technique that reduces analysis time without compromising separation performance. This technique is achieved using Active Flow Technology-Parallel Segmented Flow (AFT-PSF) columns. Extracts from cinnamon myrtle (Backhousia myrtifolia) and lemon myrtle (Backhousia citriodora) leaves were analysed via multiplexed detection using an AFT-PSF column with underivatised UV-VIS, mass spectroscopy (MS), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) derivatisation for antioxidants as detection methods. A number of antioxidant compounds were detected in the extracts of each leaf extract.

  13. Mg-ATPase activity and motility of native thick filaments isolated from the anterior byssus retractor muscle of Mytilus edulis.

    PubMed

    Yamada, A; Ishii, N; Shimmen, T; Takahashi, K

    1989-04-01

    A method for isolating native thick filaments from the anterior byssus retractor muscle (ABRM) of Mytilus edulis is described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the isolated thick filament preparation contained mainly paramyosin and myosin but almost no actin. Electron microscopy of negatively stained preparations showed that the isolated thick filaments were tapered at both ends and of various sizes, in the range 5-31 microns in length and 51-94nm in width in the central region. Central bare zones were observed in the smaller filaments, but were not clearly seen in the larger filaments. Mg-ATPase activity of the isolated thick filaments was activated by skeletal muscle F-actin in a Ca2+-dependent manner. The maximal activity was about 20 nmol min-1 mg-1 thick filaments (20 degrees C, pH7.0). Motility of the thick filaments attached to latex beads (diameter, 2 microns) was also studied using the native actin cables of the freshwater alga, Chara. In the presence of Mg-ATP and Ca2+, the beads moved along the actin cables at a maximal velocity of about 1 micron s-1. In the absence of Ca2+, almost no movement was observed. These results show that the isolated thick filaments are structurally intact and retain the essential mechanochemical characteristics of the ABRM myosin.

  14. Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor and Kunitz trypsin concentrations on the stabil...

  15. Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine modified proteins by mass spectrometry

    PubMed Central

    Chowdhury, Saiful M.; Munske, Gerhard R.; Yang, Jonathon; Zhukova, Daria; Nguen, Hamilton; Bruce, James E.

    2014-01-01

    Rationale Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies recently published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. Methods We present here a mass spectrometry-based study to evaluate and optimize the trypsin proteolysis on a guanidinated modified protein. Trypsin proteolysis was studied using different amount of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. Results We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counter parts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. Conclusion We found enzyme trypsin is active in guanidinated form of protein depending on enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on

  16. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide.

    PubMed

    Chang, Jui-Yoa

    2011-01-01

    The folding pathways of disulfide proteins vary substantially (Arolas et al., Trends Biochem Sci 31: 292-301, 2006). The diversity is mainly manifested by (a) the extent of heterogeneity of folding intermediates, (b) the extent of presence of native-like intermediates, and (c) the variation of folding kinetics. Even among structurally similar proteins, the difference can be enormous. This is demonstrated in this concise review with two structurally homologous kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor and tick anticoagulant peptide, as well as a group of cystine knot proteins. The diversity of their folding mechanisms is illustrated with two different folding techniques: (a) the conventional method of disulfide oxidation (oxidative folding), and (b) the novel method of disulfide scrambling (Chang, J Biol Chem 277: 120-126, 2002). This review also highlights the convergence of folding models concluded form the conventional conformational folding and those obtained by oxidative folding.

  17. Structural analysis of peptide fragments following the hydrolysis of bovine serum albumin by trypsin and chymotrypsin.

    PubMed

    Özyiğit, İbrahim Ethem; Akten, E Demet; Pekcan, Önder

    2016-05-01

    Peptide bond hydrolysis of bovine serum albumin (BSA) by chymotrypsin and trypsin was investigated by employing time-resolved fluorescence spectroscopy. As a fluorescent cross-linking reagent, N-(1-pyrenyl) maleimide (PM) was attached to BSA, through all free amine groups of arginine, lysine, and/or single free thiol (Cys34). Time-resolved fluorescence spectroscopy was used to monitor fluorescence decays analyzed by exponential series method to obtain the changes in lifetime distributions. After the exposure of synthesized protein substrate PM-BSA to chymotrypsin and trypsin, it is observed that each protease produced a distinct change in the lifetime distribution profile, which was attributed to distinct chemical environments created by short peptide fragments in each hydrolysate. The persistence of excimer emission at longer lifetime regions for chymotrypsin, as opposed to trypsin, suggested the presence of small-scale hydrophobic clusters that might prevent some excimers from being completely quenched. It is most likely that the formation of these clusters is due to hydrophobic end groups of peptide fragments in chymotrypsin hydrolysate. A similar hydrophobic shield was not suggested for trypsin hydrolysis, as the end groups of peptide fragments would be either arginine or lysine. Overall, in case the target protein's 3D structure is known, the structural analysis of possible excimer formation presented here can be used as a tool to explain the differences in activity between two proteases, i.e. the peak's intensity and location in the profile. Furthermore, this structural evaluation might be helpful in obtaining the optimum experimental conditions in order to generate the highest amount of PM-BSA complexes.

  18. Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).

    PubMed

    Glotzbach, Bernhard; Schmelz, Stefan; Reinwarth, Michael; Christmann, Andreas; Heinz, Dirk W; Kolmar, Harald

    2013-01-01

    In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.

  19. Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity.

    PubMed

    Reznik, Gabriel O; Yu, Yong; Tarr, George E; Cantor, Charles R

    2003-01-01

    A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.

  20. Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms.

    PubMed

    Baufreton, J; Garret, M; Dovero, S; Dufy, B; Bioulac, B; Taupignon, A

    2001-07-01

    The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABA(A) receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABA(A) receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified epsilon subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABA(A) receptors contain epsilon proteins. Applications of increasing concentrations of muscimol, a selective GABA(A) agonist, induced Cl(-) and HCO currents with an EC(50) of 5 microM. Currents induced by muscimol were fully blocked by the GABA(A) receptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an epsilon subunit-specific antibody showed that the epsilon protein was not expressed within the STN. Native subthalamic GABA(A) receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising epsilon. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABA(A) receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in

  1. Alterations in seedling vigour and antioxidant enzyme activities in Catharanthus roseus under seed priming with native diazotrophs.

    PubMed

    Karthikeyan, B; Jaleel, C A; Gopi, R; Deiveekasundaram, M

    2007-07-01

    An experiment was conducted on Catharanthus roseus to study the effect of seed treatments with native diazotrophs on its seedling growth and antioxidant enzyme activities. The treatments had significant influence on various seedling parameters. There is no significant influence on dry matter production with the diazotrophs, Azospirillum and Azotobacter. However, the vital seedling parameters such as germination percentage and vigour index were improved. Azotobacter treatment influenced maximum of 50% germination, whereas Azospirillum and Azotobacter were on par with C. roseus with respect to their vigour index. There was significant difference in the population of total diazotrophs. Azospirillum and Azotobacter between rhizosphere and non-rhizosphere soils of C. roseus had the same trend and were observed at various locations of the study. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased to a significant extent due to the treatment with diazotrophs.

  2. Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin.

    PubMed

    Patthy, András; Molnár, Tamás; Porrogi, Pálma; Naudé, Ryno; Gráf, László

    2015-01-01

    By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin-trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8 × 10(-12)M and 1.87 × 10(-12)M, respectively. The P1-P1' residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.

  3. Correlation between Antioxidant Enzyme Activity, Free Iron Content and Lipid Oxidation in Four Lines of Korean Native Chicken Meat

    PubMed Central

    Kim, Hye-Kyung; Cho, Chang-Yeon; Lee, Cheol-Koo

    2016-01-01

    This study was conducted to observe the association between antioxidant enzyme activity, free iron content and lipid oxidation of Korean native chicken (KNC) meat during refrigerated storage. Four lines of KNC (Yeonsan ogye, Hyunin black, Hoengseong yakdak and Hwangbong) were raised under similar conditions. A total of 16 roosters were randomly sampled and slaughtered at the age of 12 mon. The breast and thigh meats were stored aerobically for 10 d at 4℃. Although thigh meat had higher antioxidant enzyme activity, it was more susceptible to lipid oxidation and released more iron during storage than breast meat. Aerobic refrigerated storage for 10 d significantly decreased the activity of antioxidant enzymes and increased the amount of free iron and malondialdehyde. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were negatively correlated with lipid oxidation, whereas that of catalase was not. The amount of free iron was positively associated with lipid oxidation. We concluded that chicken line did not affect strongly on antioxidant enzyme activity and lipid oxidation in breast meat of KNC. However, the thigh meat of Hwangbong and Hyunin black had higher SOD and GSH-Px activity, respectively, and lower malondialdehyde contents than that of other chickens. SOD, GSH-Px and free iron play significant roles in meat lipid oxidation during refrigerated storage. PMID:27499663

  4. Trypsin is the culprit of multiple organ injury with severe acute pancreatitis.

    PubMed

    Sha, Huanchen; Ma, Qingyong; Jha, Rajiv Kumar

    2009-02-01

    The consistently high proportion of early deaths in patients with severe acute pancreatitis (SAP) has been associated mainly with the development of multiple organ dysfunction syndromes (MODS). So far, scholars believed that the main reasons of MODS with SAP are systemic microcirculation dysfunction and inflammatory mediator induced cascading effect on the basis of pancreas digesting itself. However, there is some special pathological phenomenon in the process of SAP which could not be explained by current theories. First, it has been evident that the pancreatic tissue bleeding and necrosis is special pathological change in pancreas autodigestive effect from digestive enzymes such as trypsin in SAP. However, we found that the liver, the lung, the intestine, the brain and the kidney have the same pathological changes in experimental animal models of SAP. Secondly, unlike the general inflammatory response, a significantly amount of bloody ascites and pleural effusion was often in patients with SAP and in experimental SAP animal models. It indicates that the vascular permeability significantly increased leading to the red blood cells extravasation. Thirdly, apart from dual blood supply, liver bears a strong compensatory function. However, liver has the highest incidence of injury in SAP when compared with other organs. In addition, we found a very interesting phenomenon after reading texts and clinical records. From the pancreatic venous drainage from the point of view, the farther the organ from the pancreas, the lower injury incidence rate observed. How to explain these mysteries? We postulate that the trypsin is the culprit of multiple organs dysfunction in SAP. The activated trypsin destroy the pancreas itself, causing pancreatic tissue bleeding and necrosis, at the same time, through venous flow it flow into the blood circulation and destroy the vascular endothelial barrier, leading to highly increased vascular permeability. So, a large number of bloody exudates

  5. Confirmation of trisomy 22 by trypsin-giemsa staining.

    PubMed Central

    Begleiter, M L; Kulkarni, P; Harris, D J

    1976-01-01

    A small-for-dates male infant with mental retardation, microcephaly, malformed ears, preauricular sinuses, epicanthal folds, micrognathia, congenital heart diseases, micropenis, and micropolygyria of the parietal and occipital lobes of the cerebral cortex was shown to have a 47,XY,+22 karyotype by trypsin-giemsa banding. Review of reported cases confirms that there may be distinctive trisomy 22 syndrome. Images PMID:138743

  6. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Trypsin test system. 862.1725 Section 862.1725 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  7. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Trypsin test system. 862.1725 Section 862.1725 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  8. Selectivity of IMAC columns in trypsin inhibitor purification.

    PubMed

    Yeomans-Reina, H; Ruiz-Manriquez, A; Wong, B R; Mansir, A T

    2001-01-01

    The properties of an adsorbent and the parameters in an adsorption process affect the resolution of chromatographic purifications. This is reflected in the elution profile, which shows the relative affinity of different proteins for a specific adsorbent. In the work presented here, elution profiles for trypsin inhibitor were used to study the effects of the concentration of trypsin inhibitor, ionic strength of the protein solution, slope of the elution gradient, and the regeneration treatment of the chromatography column on the selectivity of the adsorbent Cellufine Chelate-Cu(II)(ida). Cytochrome c was used as a reference protein. Variations in the concentrations of trypsin inhibitor and in the ionic strength of the buffered solution did not have any effects on the elution profile. On the other hand, changes in the slope of the pH gradient used for elution caused shifting of the elution peaks toward lower values of the elution volume, resulting in the best strategy to modify the elution profile of the system. Finally, using a constant slope pH gradient of elution, the variation of the selectivity of the adsorbent for trypsin inhibitor when subjected to cleaning treatments with 0.5 N NaOH was studied. Appropriate cleaning practices used in industry were followed. The adsorbent showed only a slight tendency for resolution loss in the order of 2 x 10(-4) days(-1). The results presented here show a good stability of the adsorbent when compared to other biospecific adsorbents commonly used.

  9. Native Thrombocidin-1 and Unfolded Thrombocidin-1 Exert Antimicrobial Activity via Distinct Structural Elements

    PubMed Central

    Kwakman, Paulus H. S.; Krijgsveld, Jeroen; de Boer, Leonie; Nguyen, Leonard T.; Boszhard, Laura; Vreede, Jocelyne; Dekker, Henk L.; Speijer, Dave; Drijfhout, Jan W.; te Velde, Anje A.; Crielaard, Wim; Vogel, Hans J.; Vandenbroucke-Grauls, Christina M. J. E.; Zaat, Sebastian A. J.

    2011-01-01

    Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2–3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements. PMID:22025617

  10. Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus).

    PubMed

    Zhou, Long-Zhen; Ruan, Mi-Mi; Cai, Qiu-Feng; Liu, Guang-Ming; Sun, Le-Chang; Su, Wen-Jin; Cao, Min-Jie

    2012-03-01

    A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40°C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45°C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M(-1). Apparent K(m) was 1.02 μM and k(cat) was 148 S(-1) for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5'-RACE and 3'-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.

  11. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An inter... fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute...

  12. Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn.

    PubMed

    Watthammawut, Atthaboon; Somrit, Monsicha; Asuvapongpatana, Somluk; Weerachatyanukul, Wattana

    2015-12-01

    We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm-egg penetration mechanism that is unique in this species.

  13. A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera.

    PubMed

    Gadge, Prafull P; Wagh, Sandip K; Shaikh, Faiyaz K; Tak, Rajesh D; Padul, Manohar V; Kachole, Manvendra S

    2015-11-01

    This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.

  14. Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

    PubMed

    Sjöwall, C; Zapf, J; von Löhneysen, S; Magorivska, I; Biermann, M; Janko, C; Winkler, S; Bilyy, R; Schett, G; Herrmann, M; Muñoz, L E

    2015-05-01

    In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the

  15. Cloning and characterization of trypsin- and chymotrypsin-like genes in the striped rice stem borer, Chilo suppressalis.

    PubMed

    Ge, Zhao-Yu; Wan, Pin-Jun; Han, Zhao-Jun

    2012-04-01

    Serine proteinases including trypsins and chymotrypsins play various important roles in insects, including food digestion, immune defense, and zymogen activation. Studies on insect serine proteinases could reveal their feeding preference (polyphagous and monophagous) and facilitate identification of protease inhibitors, which can be engineered for pest management. In this paper, 11 transcripts encoding trypsin- and chymotrypsin-like proteins were cloned from the striped rice stem borer, Chilo suppressalis (Walker). All the predicted proteins share high sequence similarity with known trypsin- and chymotrypsin-like proteins from either lepidopterans or dipterans, and most of the proteins have conserved motifs that are characteristics of serine proteinases. Among the 11 cloned genes, six were expressed predominantly and one exclusively in the midgut of the insect, three were expressed relatively evenly in examined tissues, and one was not expressed in either the gut or hemolymph based on RT-PCR results. The seven genes that were predominantly or exclusively expressed in the gut were also affected by feeding on different host plants. The genes that were expressed in the gut and were affected by host plants are likely to encode digestive proteinases. The identification of trypsin- and chymotrypsin-like genes in this insect species is the first step towards further comparative studies and for identification of insect-specific proteinase inhibitors, which might be engineered to protect rice plants against the striped rice stem borer, which is one of the destructive pests of rice.

  16. Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.

    PubMed

    Kecskemeti, Adam; Bako, Jozsef; Csarnovics, Istvan; Csosz, Eva; Gaspar, Attila

    2017-03-15

    Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

  17. Frequency and rates of outdoor activities, and perceptions of places to perform these activities by Native Americans and Caucasians interviewed in Tennessee.

    PubMed

    Burger, Joanna; Gochfeld, Michael; Jeitner, Christian; Pittfield, Taryn; Marchioni, Meredith

    2012-12-01

    Activity patterns and perceptions play a key role in human health risk, management, and planning. A sample of 233 people attending a Native American festival in Cookeville, Tennessee was interviewed to determine the types, percent participation, and outdoor activities rates, and their perceptions of the importance of characteristics of nuclear sites. Results indicate that: (1) a high percentage of respondents used outdoor environments, (2) they used them for consumptive (hunting, fishing), non-consumptive (hiking, walking, bird-watching), and religious/sacred activities, (3) a higher percentage of respondents engaged in non-consumptive than consumptive activities, (4) praying or meditating, communing with nature, and bird-watching had the highest uses rates (5) the environmental characteristics rated the highest were lack of radionuclides that presented a health risk, no visible smog, clean air, and unpolluted water, (6) the presence of people, buildings and roads were rated the lowest, and (7) Native Americans had higher outdoor participation rates, participated more frequently, and evaluated environmental characteristics higher than did Caucasians. This information can be used by managers to create and maintain outdoor habitats that fit the needs of local people. Planning and management require information on public policy, human needs and requirements, and human perceptions and evaluations of environmental characteristics.

  18. Frequency and rates of outdoor activities, and perceptions of places to perform these activities by Native Americans and Caucasians interviewed in Tennessee

    PubMed Central

    Gochfeld, Michael; Jeitner, Christian; Pittfield, Taryn

    2015-01-01

    Activity patterns and perceptions play a key role in human health risk, management, and planning. A survey of 233 people attending a Native American festival in Cookeville Tennessee were interviewed to determine the types, percent participation, and outdoor activities rates, and their perceptions of the importance of characteristics of sites. The indicate that: 1) a high percentage of respondents used outdoor environments, 2) they used them for consumptive (hunting, fishing), non-consumptive (hiking, walking, bird-watching), and religious/sacred activities, 3) a higher percentage of respondents engaged in non-consumptive than consumptive activities, 4) praying or meditating, communing with nature, and bird-watching had the highest uses rates 5) the environmental characteristics rated the highest were lack of radionuclides that presented a health risk, no visible smog, clean air, and unpolluted water, 6) presence of people, buildings and roads were rated the lowest and 7) Native Americans had higher participation rates, participated more frequently, and evaluated environmental characteristics higher than did Caucasians. This information can be used by managers to create and maintain outdoor habitats that fit the needs of local people. Planning and management require information on public policy, human needs and requirements, and human perceptions and evaluations of environmental characteristics. PMID:23229153

  19. Isolation and purification of trypsin inhibitors from the seeds of Abelmoschus moschatus L.

    PubMed

    Dokka, Muni Kumar; Seva, Lavanya; Davuluri, Siva Prasad

    2015-04-01

    Four trypsin inhibitors, AMTI-I, AMTI-II, AMTI-III, and AMTI-IV, have been isolated and purified to homogeneity from the seeds of Abelmoschus moschatus following ammonium sulphate fractionation, DEAE-cellulose ion exchange chromatography and gel permeation on Sephadex G-100, and their molecular weights were determined to be 22.4, 21.2, 20.8 and 20.2 kDa respectively by SDS-PAGE. While all the four inhibitors were very active against bovine trypsin, two of them (AMTI-III and AMTI-IV) showed moderate activity towards bovine chymotrypsin. AMTI-I and AMTI-II were found to be glycoproteins with neutral sugar content of 2.8 and 4 %, respectively, and all the four inhibitors were devoid of free sulphhydryl groups. The inhibitors were quite stable up to 80 °C for 10 min and were not affected at alkaline as well as acidic conditions tested. Treating them with 8 M urea and 1 % SDS for 24 h at room temperature did not result in any loss of their antitryptic activities. However, they lost considerable antitryptic activity when treated with 6 M guanidine hydrochloride. Activities of the inhibitors were unaffected even after their reduction with DTT suggesting that disulphide bonds are not needed for their inhibitory activities.

  20. Superior Temporal Activation as a Function of Linguistic Knowledge: Insights from Deaf Native Signers Who Speechread

    ERIC Educational Resources Information Center

    Capek, Cheryl M.; Woll, Bencie; MacSweeney, Mairead; Waters, Dafydd; McGuire, Philip K.; David, Anthony S.; Brammer, Michael J.; Campbell, Ruth

    2010-01-01

    Studies of spoken and signed language processing reliably show involvement of the posterior superior temporal cortex. This region is also reliably activated by observation of meaningless oral and manual actions. In this study we directly compared the extent to which activation in posterior superior temporal cortex is modulated by linguistic…

  1. Assessment of Anti-Quorum Sensing Activity for Some Ornamental and Medicinal Plants Native to Egypt

    PubMed Central

    Zaki, Ahmed A.; Shaaban, Mona I.; Hashish, Nadia E.; Amer, Mohamed A.; Lahloub, Mohamed-Farid

    2013-01-01

    This study investigated the effects of some plant extracts on the bacterial communication system, expressed as quorum sensing (QS) activity. Quorum sensing has a directly proportional effect on the amount of certain compounds, such as pigments, produced by the bacteria. Alcohol extracts of 23 ornamental and medicinal plants were tested for anti-QS activity by the Chromobacterium violaceum assay using the agar cup diffusion method. The screening revealed the anti-QS activity of six plants; namely the leaves of Adhatoda vasica Nees, Bauhinia purpurea L., Lantana camara L., Myoporum laetum G. Forst.; the fruits of Piper longum L.; and the aerial parts of Taraxacum officinale F.H. Wigg. PMID:23641343

  2. [Effect of heliogeophysical factors on epizootic activity of a native focus of visceral leishmaniasis].

    PubMed

    Oiburivsjuĭ, E N

    2003-01-01

    Visceral leishmaniasis (VL) morbidity in the population of the south-eastern Turkmenistan and the epizootic activity of a natural focus were studied. A definite cyclic recurrence of the processes occurring in the natural focus of VL, which was associated with solar activity changes was found. The regularities found make it possible to predict the increase in the epizootic tension of a natural focus of VL in this region and the incidence of this disease among the population in 2002-2004.

  3. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    Golbeck, J H; Cammarata, K V

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  4. Selective release of excreted DNA sequences from phytohemagglutinin-stimulated human peripheral blood lymphocytes. Effects of trypsin and divalent cations.

    PubMed Central

    Distelhorst, C W; Cramer, K; Rogers, J C

    1978-01-01

    We studied the synthesis of excreted DNA sequences and their release from phytohemagglutinin-stimulated human peripheral blood lymphocytes under conditions permitting optimal cell growth. Cells were labeled by constant exposure to low specific activity [3H]thymidine. Excreted DNA sequences were synthesized during the period of logarithmic cell growth and moved slowly from the high molecular weight chromosomal DNA fraction into the low molecular weight cell DNA fraction (Hirt supernate) from which they could be specifically released by treating the cells briefly with small amounts of various proteases; 1 microgram/ml trypsin for 5 min was optimal. On day 5 of culture, 13.3 +/- 6.9% of the total cellular acid-precipitable [3H]thymidine was released by this treatment. Trypsin-induced release was partially and reversibly inhibited by incubating the cells for 16 h with 5 mM dibutyryl-cyclic AMP. Cells incubated in the absence of divalent cations spontaneously released this Hirt supernatant DNA; after maximal release had occurred under these circumstances, additional trypsin treatment caused no further release of DNA. Trypsin-induced DNA release could be completely and reversibly inhibited by incubating the cells in the presence of 10 mM calcium. Trypsin-released DNA was isolated and analyzed by reassociation kinetics. A major component, representing 54% of the DNA, reassociated with a C0t1/2 of 68 mol.s/liter (the value at which DNA association is 50% complete). The reassociation of this DNA was studied in the presence of an excess of DNA isolated from stimulated lymphocytes on day 3 in culture, and in the presence of an excess of resting lymphocyte DNA. The high molecular weight fraction of day-3 cell DNA contained three times more copies of the trypsin-released DNA major component as compared to resting lymphocyte DNA. Hirt supernatant DNA isolated from day-5 stimulated lymphocytes reassociated in an intermediate component representing 34% of the DNA with a Cot1/2 of

  5. Urinary Trypsin Inhibitor Ameliorates Seawater Immersion-Induced Intestinal Mucosa Injury via Antioxidation, Modulation of NF-κB Activity, and Its Related Cytokines in Rats with Open Abdominal Injury

    PubMed Central

    Zhang, Xing Jian; Wang, Ya Li; Zhou, Song; Xue, Xiaojun; Liu, Qiang; Zhang, Wen Hua; Zheng, Jun

    2014-01-01

    Objective. To investigate the role of oxidative stress, NF-κB activity, and its related cytokines in the pathogenesis of seawater immersion after open abdominal injury (SI-OAI) and whether UTI treatment can attenuate SI-OAI induced IMI. Methods. Wistar rats were randomly divided into three groups: C group, S group, and U group. The rats in C group only suffered from anesthesia and surgical operation, whereas the rats in S group and U group received caudal vein injection of normal saline without/with 50,000 U/kg body weight of UTI. The activities of TNF-α, IL-6, SOD, MDA, ROS, NF-κB, and IκB-β were monitored by ELISA, biochemical methods, EMSA, and Western blot, respectively. Results. The plasma inflammatory mediators and the contents of MDA, ROS, and NF-κB in intestine as well as the pathological scores in ileal mucosa were significantly increased in rats after SI-OAI, accompanied by a reduction in SOD activities and IκB-β levels. UTI treatment significantly attenuated intestinal histopathological changes with evidence of a decrease in all of the parameters, except for upregulation of the levels of SOD and IκB-β protein. Conclusion. UTI can attenuate SI-OAI induced IMI via inhibition of NF-κB activity, subsequently inhibiting the expression of inflammatory cytokines and by combating oxidative stress. PMID:25210512

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

    SciTech Connect

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

    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.

  7. Adsorption of Pb(II) ions from aqueous solution by native and activated bentonite: kinetic, equilibrium and thermodynamic study.

    PubMed

    Kul, Ali Riza; Koyuncu, Hülya

    2010-07-15

    In this study, the adsorption kinetics, equilibrium and thermodynamics of Pb(II) ions on native (NB) and acid activated (AAB) bentonites were examined. The specific surface areas, pore size and pore-size distributions of the samples were fully characterized. The adsorption efficiency of Pb(II) onto the NB and AAB was increased with increasing temperature. The kinetics of adsorption of Pb(II) ions was discussed using three kinetic models, the pseudo-first-order, the pseudo-second-order and the intra-particle diffusion model. The experimental data fitted very well the pseudo-second-order kinetic model. The initial sorption rate and the activation energy were also calculated. The activation energy of the sorption was calculated as 16.51 and 13.66 kJ mol(-1) for NB and AAB, respectively. Experimental results were also analysed by the Langmuir, Freundlich and Dubinin-Redushkevich (D-R) isotherm equations at different temperatures. R(L) separation factor for Langmuir and the n value for Freundlich isotherm show that Pb(II) ions are favorably adsorbed by NB and AAB. Thermodynamic quantities such as Gibbs free energy (DeltaG), the enthalpy (DeltaH) and the entropy change of sorption (DeltaS) were determined as about -5.06, 10.29 and 0.017 kJ mol(-1) K(-1), respectively for AAB. It was shown that the sorption processes were an endothermic reactions, controlled by physical mechanisms and spontaneously.

  8. Effect of the native polysaccharide of cashew-nut tree gum exudate on murine peritoneal macrophage modulatory activities.

    PubMed

    Yamassaki, F T; Lenzi, R M; Campestrini, L H; Bovo, F; Seyfried, M; Soldera-Silva, A; Stevan-Hancke, F R; Zawadzki-Baggio, S F; Pettolino, F A; Bacic, A; Maurer, J B B

    2015-07-10

    The native polysaccharide of cashew-nut tree gum exudate (CNTG) and its arabinogalactan-protein component (CNTG-AGP) were tested by using immuno-stimulant and anti-inflammatory in vitro assays of murine peritoneal macrophage activities. In the assay for immuno-stimulant activity (without previous treatment with lipopolysaccharide; LPS), CNTG increased the production of interleukin (IL)-10 and both CNTG and CNTG-AGP decreased the concentrations of IL6. When the macrophages were incubated in the presence of LPS and CNTG a decrease in the levels of nitric oxide (NO(·)) and IFN-γ was observed. The results could explain the popular use of CNTG as an anti-inflammatory. In addition, CNTG is the main component of the cashew-nut tree gum exudate, which has been considered a versatile polymer with potential pharmaceutical and food industry applications. These data may contribute to the study of the immunomodulation activity of plant polysaccharides, as well as encourage future experiments in the field of cashew-nut tree gum exudate applications.

  9. Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres.

    PubMed

    Lin, Shuang; Yao, Guoping; Qi, Dawei; Li, Yan; Deng, Chunhui; Yang, Pengyuan; Zhang, Xiangmin

    2008-05-15

    A fast and efficient proteolysis approach of microwave-assisted protein digestion was developed by using trypsin-immobilized magnetic silica (MS) microspheres. In the work, immobilization of the enzyme onto MS microspheres was very simple and only through a one-step reaction with 3-glycidoxypropyltrimethoxysilane (GLYMO) which provides the epoxy group as a reactive spacer. Considering that the magnetic particles are excellent microwave absorbers, we developed a novel microwave-assisted digestion method based on the easily prepared trypsin-immobilized MS microspheres. This novel digestion method combined the advantages of immobilized trypsin and the rapid-fashion of microwave-assisted digestion, which resulted in high digestion efficiency. BSA and myoglobin were used as model proteins to optimize the conditions of this method. Peptide fragments produced in 15 s could be confidently identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Equivalent or better digestion efficiency was observed comparing to current in-solution digestion. Besides, because of the unique magnetic responsivity, the immobilized trypsin can be isolated easily with the help of an external magnet and thus used repeatedly. High activity was obtained even after seven runs of the trypsin-immobilized MS microspheres. To further verify its efficiency in proteome analysis, one reversed-phase liquid chromatography (RPLC) fraction of rat liver extract was applied. After 15 s incubation, 16 totally unique peptides corresponding to two proteins were identified. Finally, the rat liver sample was used to evaluate its worth for the application. With analysis by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS), comparable digestion efficiency was observed with typical in-solution digestion but the incubation time was largely shortened. This new microwave-assisted digestion method will hasten the application of the proteome

  10. Structural characterization of native autoinducing peptides and abiotic analogues reveals key features essential for activation and inhibition of an AgrC quorum sensing receptor in Staphylococcus aureus.

    PubMed

    Tal-Gan, Yftah; Ivancic, Monika; Cornilescu, Gabriel; Cornilescu, Claudia C; Blackwell, Helen E

    2013-12-11

    Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I-IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible antivirulence strategies for the treatment of infection. We recently reported a set of analogues of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogues that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I-IV) and several AIP-III analogues with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic "knob" essential for both activation and inhibition and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus , and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

  11. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

    PubMed Central

    Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

    1992-01-01

    Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

  12. [Serological tests of functional activity of the digestive system (gastrin, pepsinogen-I, trypsin), general IgE and serum cortisol levels in children with hepatitis A and B].

    PubMed

    Kalagina, L S; Pavlov, Ch S; Fomin, Iu A

    2013-01-01

    The mild form of hepatitis A and B with children is attended by a functional activity of pancreatic gland (tripsin), mucous coats of stomach and duodenum (gastrin) which permits to consider them as a factor of the risk of digestive organs combined pathology starting with the disease acuity. Differences in gastrin levels with children depending on hepatitis etiology were specified. Highest levels of gastrin were observed with persons suffering from hepatitis B.

  13. Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The multifunctional arthropod insect kinins share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects. Compounds with similar biological activity cou...

  14. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  15. Community Health Representatives: A Valuable Resource for Providing Coronary Heart Disease Health Education Activities for Native Americans.

    ERIC Educational Resources Information Center

    Cleaver, Vicki L.

    1989-01-01

    This article addresses select health issues of Native Americans, emphasizing coronary heart disease (CHD). The link between lifestyle and CHD is discussed. CHD risk data from a study of 67 Community Health Representatives is presented, and the role these paraprofessionals can play in health education among Native Americans is discussed. (IAH)

  16. [Conformation of trypsin molecules in aqueous solutions containing 2-chloroethanol].

    PubMed

    Kushner, V P

    1980-01-01

    Changes in the macromolecular parameters of trypsin in the presence of 2-chloroethanol in aqueous solutions have been studied by means of optical and hydrodynamic methods. At temperature--dependent volume fraction of 2-chloroethanol in solution upsilon t < 0.30 the globular structure of trypsin is destroyed but the regularity of polypeptide chains within the limits of secondary structure is maintained. At 0.30 < upsilon < 0.80 the solvation envelope of macromolecules is kept constant mainly owing to hydration, but the solubilization takes place only at upsilon < 0.30. At upsilon < 0.80 spiralization sharply increases and reaches in pure 2-chloroethanol its maximum value (50%). The intrinsic viscosity moreover reaches only half the whole value [eta]coil--[eta]glob.

  17. Biostable Agonists that Match or Exceed Activity of Native Insect Kinins on Recombinant Arthropod GPCRs

    DTIC Science & Technology

    2009-01-01

    diuresis in many species of insects. Compounds with similar biological activity could be exploited for the control of arthropod pest populations such as...species, insect kinins stimulate hindgut contractions, diuresis , digestive enzyme release and probably inhibit larval weight gain (Holman et al., 1990...conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in

  18. Batch and dynamic sorption of Ni(II) ions by activated carbon based on a native lignocellulosic precursor.

    PubMed

    Nabarlatz, Debora; de Celis, Jorge; Bonelli, Pablo; Cukierman, Ana Lea

    2012-04-30

    Vinal-derived Activated Carbon (VAC) developed by phosphoric acid activation of sawdust from Prosopis ruscifolia native wood was tested for the adsorption of Ni(II) ions from dilute solutions in both batch and dynamic modes, comparing it with a Commercial Activated Carbon (CAC). Batch experiments were performed to determine adsorption kinetics and equilibrium isotherms for both carbons. It was possible to remove near 6.55 mg Ni g(-1) VAC and 7.65 mg Ni g(-1) CAC after 5 h and 10 h contact time, respectively. A pseudo second order equation fitted well with the kinetics of the process, and Langmuir adsorption model was used to adjust the experimental results concerning the adsorption isotherm. The parameters obtained indicate a stronger interaction between sorbent and sorbate for VAC (K = 26.56 L mmol(-1)) than for CAC (K = 19.54 L mmol(-1)). Continuous experiments were performed in a fixed-bed column packed with the investigated carbons, evaluating the influence of operational parameters such as flow rate, bed height and feed concentration on the breakthrough curves obtained. The breakthrough occurred more slowly for low concentrations of the metal ion in the feed, low flow rates and high bed height. The breakthrough curves were properly represented by Hall's model for both carbon types. Regeneration of the vinal activated carbon in column was tested, obtaining the same breakthrough curve in a new cycle of use. Finally, vinal-derived activated carbon can effectively be used to treat wastewater having until 30 ppm Ni(II).

  19. Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors.

    PubMed

    Steppuhn, Anke; Baldwin, Ian T

    2007-06-01

    Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua, a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.

  20. Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54(nrb)) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

  1. Crystallographic and kinetic evidence of allostery in a trypsin-like protease.

    PubMed

    Niu, Weiling; Chen, Zhiwei; Gandhi, Prafull S; Vogt, Austin D; Pozzi, Nicola; Pelc, Leslie A; Zapata, Fatima; Di Cera, Enrico

    2011-07-26

    Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing a displacement of 6.6 Å of the entire 215-217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.

  2. Trypsin resistance of a decapeptide KISS1R agonist containing an Nω-methylarginine substitution.

    PubMed

    Asami, Taiji; Nishizawa, Naoki; Ishibashi, Yoshihiro; Nishibori, Kimiko; Horikoshi, Yasuko; Matsumoto, Hirokazu; Ohtaki, Tetsuya; Kitada, Chieko

    2012-10-15

    Metastin/kisspeptin is an amidated peptide with 54 amino acid residues isolated from human placental tissues as a ligand of the orphan G-protein-coupled receptor KISS1R that is expressed throughout the central nervous system and in a variety of endocrine and gonadal tissues. Compared to the full-length metastin protein, the N-terminal truncated peptide metastin(45-54) has 3-10 times higher receptor affinity and enhanced ability to increase intracellular calcium concentration which is essential for activation of protein kinases involved in intracellular signaling in a number of pathways that affect reproduction and cell migration. However, metastin(45-54) is rapidly inactivated in serum. In this study, we designed and synthesized a number of metastin(45-54) analogs and evaluated their agonistic activity and trypsin resistance. Among analogs with substitutions of arginine at position 53, N(ω)(-)methylarginine analog 8 showed 3-fold more potent agonistic activity compared with metastin(45-54). Furthermore, analog 8 was shown to resist trypsin cleavage between positions 53 and 54. This substitution may be useful in the development of other Arg-containing peptides for which the avoidance of cleavage is desired.

  3. In Vivo Efficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

    PubMed Central

    Malik, U.; Silva, O. N.; Fensterseifer, I. C. M.; Chan, L. Y.; Clark, R. J.; Franco, O. L.; Daly, N. L.

    2015-01-01

    Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen. PMID:25624332

  4. Extracellular trypsin-like proteases produced by Cordyceps militaris.

    PubMed

    Hattori, Maki; Isomura, Shigeki; Yokoyama, Eiji; Ujita, Minoru; Hara, Akira

    2005-12-01

    A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.

  5. Role of native and exotic woody vegetation in soil restoration in active gully systems (southern Ecuador)

    NASA Astrophysics Data System (ADS)

    Borja Ramon, Pablo; Alvarado Moncayo, Dario; Vanacker, Veerle; Cisneros, Pedro; Molina, Armando; Govers, Gerard

    2015-04-01

    Revegetation projects in degraded lands have the potential to recover essential soil functions. If vegetation restoration is combined with bioengineering techniques, such as the construction of retention dams in active gully systems, soil restoration could be enhanced. One important aspect of this process is the role of vegetation on restoration of soil chemical and physical properties. There is currently a lack of knowledge on the potential of soil restoration in active badland systems, as most studies have concentrated on the direct and visible effect of revegetation on erosion control. The aim of this study is to evaluate the role of revegetation and bioengineering works on the restoration of soil physical and chemical properties. The analyses are realized in a highly degraded area of 3 km2, located in the lower part of the Loreto catchment (Southern Ecuadorian Andes). First, the soil physical and/or chemical parameters that are most sensitive to track environmental change were evaluated. Second, the role of vegetation on soil restoration was quantified. . Soil samples were taken in sites with different vegetation cover, land use and physiographic position. The following physical and chemical parameters were measured: volumetric water content (θsat, θact), bulk density, pH, texture, organic matter, C and N content. Our first results do not show a clear relationship between volumetric water content at saturation (θsat), bulk density, or C content. The saturation water content does not vary significantly between different sites, or land use types. However, significant differences are found between sites at different stages of restoration; and this for most chemical and physical soil properties. Vegetation cover (%) appears to exert a strong control on the C content in the mineral soils. The highest C values are found in soils of forest plantations with Eucalyptus and Pinus species. These plantations are located in areas that were previously affected by active

  6. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrate...

  7. Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion.

    PubMed

    Jiang, Bo; Yang, Kaiguang; Zhao, Qun; Wu, Qi; Liang, Zhen; Zhang, Lihua; Peng, Xiaojun; Zhang, Yukui

    2012-09-07

    In this paper, magnetic Fe₃O₄ nanoparticles modified graphene oxide nanocomposites (GO-CO-NH-Fe₃O₄) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe₃O₄ and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO-CO-NH-Fe₃O₄ as a novel substrate, trypsin was immobilized via π-π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg. Since GO-CO-NH-Fe₃O₄ worked as not only support for enzyme immobilization, but also as an excellent microwave irradiation absorber, the digestion efficiency could be further improved with microwave assistance. By such an immobilized enzymatic reactor (IMER), standard proteins could be efficiently digested within 15 s, with sequence coverages comparable or better than those obtained by conventional in-solution digestion (12 h). Since trypsin was immobilized under mild conditions, the enzymatic activity of IMER preserved at least for a month. In addition, due to the good hydrophilicity of GO, no peptide residue was observed in the sequent digestion of bovine serum albumin and myoglobin. To further confirm the efficiency of such an IMER for proteome analysis, it was applied to digest proteins extracted from rat liver, followed by nanoRPLC-ESI-MS/MS analysis. With only 5 min microwave-assisted digestion, in 3 parallel runs, totally 456 protein groups were identified, comparable to that obtained by 12 h in-solution digestion, indicating the great potential of IMERs with GO-CO-NH-Fe₃O₄ as the support for high throughput proteome study.

  8. Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

    SciTech Connect

    Galakatos, N.G.; Walsh, C.T.

    1987-12-15

    Native DadB and Alr alanine racemases (M/sub r/ 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by ..cap alpha..-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of M/sub r/ 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator ..beta..-chloro-(/sup 14/C)-D-alanine, and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB/sup 3/H/sub 4/-reduced DadB holoenzyme is resistant to ..cap alpha..-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.

  9. Amino acid sequence of homologous rat atrial peptides: natriuretic activity of native and synthetic forms.

    PubMed Central

    Seidah, N G; Lazure, C; Chrétien, M; Thibault, G; Garcia, R; Cantin, M; Genest, J; Nutt, R F; Brady, S F; Lyle, T A

    1984-01-01

    A substance called atrial natriuretic factor (ANF), localized in secretory granules of atrial cardiocytes, was isolated as four homologous natriuretic peptides from homogenates of rat atria. The complete sequence of the longest form showed that it is composed of 33 amino acids. The three other shorter forms (2-33, 3-33, and 8-33) represent amino-terminally truncated versions of the 33 amino acid parent molecule as shown by analysis of sequence, amino acid composition, or both. The proposed primary structure agrees entirely with the amino acid composition and reveals no significant sequence homology with any known protein or segment of protein. The short form ANF-(8-33) was synthesized by a multi-fragment condensation approach and the synthetic product was shown to exhibit specific activity comparable to that of the natural ANF-(3-33). PMID:6232612

  10. Native Intelligence

    ERIC Educational Resources Information Center

    Seven, Richard

    2006-01-01

    Amid concerns from tribal leaders that No Child Left Behind testing is squeezing out electives that have traditionally covered their history and cultures, an ambitious brace of programs is making Native America part of the core curriculum at David Wolfle Elementary School and other schools in the western Washington State. By tapping into…

  11. U.S. Geological Survey activities related to American Indians and Alaska Natives: Fiscal years 2007 and 2008

    USGS Publications Warehouse

    Marcus, Susan M.

    2010-01-01

    In the late 1800s, John Wesley Powell, the second director of the U.S. Geological Survey (USGS), followed his interest in the tribes of the Great Basin and Colorado Plateau and studied their cultures, languages, and surroundings. From that early time, the USGS has recognized the importance of Native knowledge and living in harmony with nature as complements to the USGS mission to better understand the Earth. Combining traditional ecological knowledge with empirical studies allows the USGS and Native American governments, organizations, and people to increase their mutual understanding and respect for this land. The USGS is the earth and natural science bureau within the U.S. Department of the Interior (DOI) and is not responsible for regulations or land management. Climate change is a major current issue affecting Native lives and traditions throughout the United States. Climate projections for the coming century indicate an increasing probability for more frequent and more severe droughts in the Southwest, including the Navajo Nation. Erosion has claimed Native homes in Alaska. Fish have become inedible due to diseases that turn their flesh mushy. Native people who rely on or who are culturally sustained by hunting, fishing, and using local plants are living with climate change now. The traditional knowledge of Native peoples enriches and confirms the work of USGS scientists. The results are truly synergistic-greater than the sum of their parts. Traditional ecological knowledge is respected and increasingly used in USGS studies-when the holders of that knowledge choose to share it. The USGS respects the rights of Native people to maintain their patrimony of traditional ecological knowledge. The USGS studies can help Tribes, Native organizations, and natural resource professionals manage Native lands and resources with the best available unbiased data and information that can be added to their traditional knowledge. Wise Native leaders have noted that traditional

  12. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

    PubMed Central

    2010-01-01

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity. "One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity." Josiah Willard Gibbs (1839-1903) PMID:20534114

  13. Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

    PubMed

    Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

    2014-03-01

    Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

  14. Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis.

    PubMed

    Yu, X Christopher; Joe, Koman; Zhang, Yu; Adriano, Andrea; Wang, Yaning; Gazzano-Santoro, Helene; Keck, Rodney G; Deperalta, Galahad; Ling, Victor

    2011-08-01

    We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.

  15. Comparative studies on the thermal properties of a trypsin-like protease intwo hermit crabs

    NASA Astrophysics Data System (ADS)

    Dittrich, Birgit

    1992-03-01

    The thermal characteristics of a trypsin-like protease were surveyed comparatively in two hermit crabs, Pagurus bernhardus (Linné) 1758 from the German Bight, and Clibanarius striolatus Dana 1852 from the Western Indo-Pacific. In both enzymes, activity is maximal at a temperature around 50°C. Compared with Pagurus, the protease in Clibanarius is characterized by a considerably higher stability at elevated temperatures. Furthermore, the latter is less inhibited by two specific trypsin inhibitors. On an energetical level, distinct differences between the species are displayed. In both species, Km is strongly affected by temperature; lowest Km values do not coincide with the mean environmental temperature. The affinity of Pagurus protease for substrate at 40°C is about 17 times that at 0°C; in Clibanarius this factor amounts only to 4.4. At temperatures >10°C, activation energy in the tropical species Clibanarius is distinctly higher (28.3 kJ·mol-1) than in the boreal species Pagurus (20.0 kJ·mol-1).

  16. Comparative effects of ohmic, induction cooker, and electric stove heating on soymilk trypsin inhibitor inactivation.

    PubMed

    Lu, Lu; Zhao, Luping; Zhang, Caimeng; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-03-01

    During thermal treatment of soymilk, a rapid incorporation of Kunitz trypsin inhibitor (KTI) into protein aggregates by covalent (disulfide bond, SS) and/or noncovalent interactions with other proteins is responsible for its fast inactivation of trypsin inhibitor activity (TIA). In contrast, the slow cleavage of a single Bowman-Birk inhibitor (BBI) peptide bond is responsible for its slow inactivation of TIA and chymotrypsin inhibitor activity (CIA). In this study, the effects of Ohmic heating (220 V, 50 Hz) on soymilk TIA and CIA inactivation were examined and compared to induction cooker and electric stove heating with similar thermal histories. It was found that: (1) TIA and CIA inactivation was slower from 0 to 3 min, and faster after 3 min as compared to induction cooker and electric stove. (2) The thiol (SH) loss rate was slower from 0 to 3 min, and similar to induction cooker and electric stove after 3 min. (3) Ohmic heating slightly increased protein aggregate formation. (4) In addition to the cleavage of one BBI peptide bond, an additional reaction might occur to enhance BBI inactivation. (5) Ohmic heating was more energy-efficient for TIA and CIA inactivation. (6) TIA and CIA inactivation was accelerated with increasing electric voltage (110, 165, and 220 V) of Ohmic heating. It is likely that the enhanced inactivation of TIA by Ohmic heating is due to its combined electrochemical and thermal effects.

  17. [Low-molecular cytolysins and trypsin inhibitors from sea anemone Radianthus macrodactylus. Isolation and partial characterization].

    PubMed

    Zykova, T A; Monastyrnaia, M M; Apalikova, O V; Shvets, T V; Kozlovskaia, E P

    1998-07-01

    Two low-molecular cytolytic toxins (RmI and RmII) and four trypsin inhibitors were isolated from the aqueous extract of sea anemone Radianthus macrodactylus. The method of isolation involved precipitation with acetone, gel filtration on acrylex P-4, ion-exchange chromatography on CM-32 cellulose, affinity chromatography on trypsin-binding sepharose 4B, ion exchange chromatography on an Ultrapore TSK CM-3SW column, and reversed phase HPLC on a Silasorb C18 column. RmI, RmII, and JnI inhibitor displayed molecular masses 5100, 6100, and 7100 Da, respectively, when subjected to SDS-PAGE. The isoelectric points were 9.2 and 9.3 for RmI and RmII, respectively. The amino acid composition and N-terminal amino acid residue (glycine) were determined for RmI, RmII, and JnI. Both proteins were nontoxic to mice and crabs. Hemolytic activity was determined to be 25 and 20 HU/mg for RmI and RmII, respectively, and their action on erythrocyte membrane was not inhibited by exogenous sphingomyelin. RmI and RmII exhibited antihistamine activity.

  18. Conferring biological activity to native spider silk: A biofunctionalized protein-based microfiber.

    PubMed

    Wu, Hsuan-Chen; Quan, David N; Tsao, Chen-Yu; Liu, Yi; Terrell, Jessica L; Luo, Xiaolong; Yang, Jen-Chang; Payne, Gregory F; Bentley, William E

    2017-01-01

    Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

  19. The antiprotozoal activity of sixteen asteraceae species native to Sudan and bioactivity-guided isolation of xanthanolides from Xanthium brasilicum.

    PubMed

    Nour, Amal M M; Khalid, Sami A; Kaiser, Marcel; Brun, Reto; Abdallah, Wai'l E; Schmidt, Thomas J

    2009-10-01

    In vitro screening of the dichloromethane extracts of 16 Asteraceae species native to Sudan for activity against major protozoan pathogens revealed that a Xanthium brasilicum Vell. [syn. X. strumarium var. brasilicum (Vell.) Baker in Mart.] extract was the most active against Trypanosoma brucei rhodesiense, the etiological agent of East African human trypanosomiasis (IC(50) = 0.1 microg/mL). This plant extract also exhibited noticeable activities against T. cruzi (Chagas disease), Leishmania donovani (Kala-Azar) as well as Plasmodium falciparum (Malaria tropica). Bioactivity-guided fractionation resulted in the isolation of four bioactive sesquiterpene lactones (STL) of the xanthanolide series (4,5-seco-guaianolide-type). They were identified by spectroscopic means as 8-epixanthatin (1), 8-epixanthatin 1beta,5beta-epoxide (2), and as the dimers pungiolide A (4) as well as pungiolide B (5). Two further modified xanthanolide sesquiterpene lactones, xanthipungolide (3) and 4,15-dinor-1,11(13)-xanthadiene-3,5beta:12,8beta-diolide (6) were isolated. While xanthipungolide turned out to be inactive against the tested parasites, the dinor-xanthanlide showed significant activity against T. brucei rhodesiense and L. donovani. All isolated compounds were previously known from other Xanthium species but this is the first report on their occurrence in X. brasilicum, and, most notably, on their antiprotozoal activity. As the most active single compound from this extract, 8-epixanthatin 1beta,5beta-epoxide showed IC(50) values of 0.09, 2.95, 0.16 and 1.71 microg/mL (0.33, 11.3, 0.6 and 6.5 microM) against T. brucei rhodesiense, T. cruzi, L. donovani and P. falciparum, respectively, while its cytotoxicity against rat myoblast cells used as control was determined at 5.8 microg/mL (22.1 microM). Besides assessment of their antiprotozoal activity, the structural assignments for the dimeric xanthanolides pungiolide A and B were reinvestigated and fully established.

  20. Comparative resistance and resilience of soil microbial communities and enzyme activities in adjacent native forest and agricultural soils.

    PubMed

    Chaer, Guilherme; Fernandes, Marcelo; Myrold, David; Bottomley, Peter

    2009-08-01

    Degradation of soil properties following deforestation and long-term soil cultivation may lead to decreases in soil microbial diversity and functional stability. In this study, we investigated the differences in the stability (resistance and resilience) of microbial community composition and enzyme activities in adjacent soils under either native tropical forest (FST) or in agricultural cropping use for 14 years (AGR). Mineral soil samples (0 to 5 cm) from both areas were incubated at 40 degrees C, 50 degrees C, 60 degrees C, or 70 degrees C for 15 min in order to successively reduce the microbial biomass. Three and 30 days after the heat shocks, fluorescein diacetate (FDA) hydrolysis, cellulase and laccase activities, and phospholipid-derived fatty acids-based microbial community composition were measured. Microbial biomass was reduced up to 25% in both soils 3 days after the heat shocks. The higher initial values of microbial biomass, enzyme activity, total and particulate soil organic carbon, and aggregate stability in the FST soil coincided with higher enzymatic stability after heat shocks. FDA hydrolysis activity was less affected (more resistance) and cellulase and laccase activities recovered more rapidly (more resilience) in the FST soil relative to the AGR counterpart. In the AGR soil, laccase activity did not show resilience to any heat shock level up to 30 days after the disturbance. Within each soil type, the microbial community composition did not differ between heat shock and control samples at day 3. However, at day 30, FST soil samples treated at 60 degrees C and 70 degrees C contained a microbial community significantly different from the control and with lower biomass regardless of high enzyme resilience. Results of this study show that deforestation followed by long-term cultivation changed microbial community composition and had differential effects on microbial functional stability. Both soils displayed similar resilience to FDA hydrolysis, a

  1. Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus.

    PubMed

    Perdomo-Morales, R; Montero-Alejo, V; Perera, E; Pardo-Ruiz, Z; Alonso-Jiménez, E

    2007-12-01

    The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.

  2. Flavor precursors and sensory-active sulfur compounds in alliaceae species native to South Africa and South America.

    PubMed

    Kubec, Roman; Krejčová, Petra; Mansur, Leví; García, Nicolás

    2013-02-13

    Profiles of S-substituted cysteine flavor precursors were determined in 42 Alliaceae species native to South Africa and South America. It was found that the pool of cysteine derivatives present in these plants is remarkably very simple, with S-((methylthio)methyl)cysteine 4-oxide (marasmin) being the principal flavor precursor, typically accounting for 93-100% of the pool. Out of the other cysteine derivatives, only minor quantities of methiin were present in some species. The marasmin-derived thiosulfinate marasmicin (2,4,5,7-tetrathiaoctane 4-oxide), a major sensory-active compound of the freshly disrupted plants, was isolated, and its organoleptic properties were evaluated. Furthermore, sulfur-containing volatiles formed upon boiling of these alliaceous species were studied by GC-MS. The profile of the volatiles formed was relatively simple, with 2,3,5-trithiahexane and 2,4,5,7-tetrathiaoctane being the major components. Despite the traditional belief, ingestion of the marasmin-rich plants was always accompanied by development of a strong "garlic breath". We believe that especially several Tulbaghia species deserve to attract much greater attention from the food industry thanks to their pungent garlicky taste and unusual yet pleasant alliaceous smell.

  3. Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry.

    PubMed

    Ohla, Stefan; Schulze, Philipp; Fritzsche, Stefanie; Belder, Detlev

    2011-02-01

    In the present work, we report on a rapid and straightforward approach for the determination of biologically active compounds in bananas applying microchip electrophoresis (MCE). For this purpose, we applied label-free detection utilizing deep UV fluorescence detection with excitation at 266 nm. Using this approach, we could identify dopamine and serotonin, their precursors tryptophan and tyrosine and also the isoquinoline alkaloid salsolinol in less than 1 min. In bananas, after 10 days of ripening, we additionally found the compound levodopa which is a metabolite of the tyrosine pathway. Quantitative analysis of extracts by external calibration revealed concentrations of serotonin, tryptophan, and tyrosine from 2.7 to 7.6 μg/mL with relative standard deviations of less than 3.5%. The corresponding calibration plots showed good linearity with correlation coefficients higher than 0.985. For reliable peak assignment, the compounds were also analyzed by coupling chip electrophoresis with mass spectrometry. This paper demonstrates exemplarily the applicability of MCE with native fluorescence detection for rapid analysis of natural compounds in fruits and reveals the potential of chip-based separation systems for the analysis of complex mixtures.

  4. Covalent immobilization of mixed proteases, trypsin and chymotrypsin, onto modified polyvinyl chloride microspheres.

    PubMed

    Li, Dong-Fang; Ding, Hao-Chen; Zhou, Tao

    2013-11-06

    A commercially available trypsin-chymotrypsin mixture was covalently immobilized onto modified polyvinyl chloride (PVC) microspheres, which were activated by the subsequent treatment of PVC microspheres with ethylenediamine and glutaraldehyde. The immobilized mixed protease was characterized by FT-IR and SEM analyses. Immobilization conditions were optimized by Box-Behnken design and the response surface method. The activity of the immobilized mixed protease prepared under optimal conditions (pH 6.6, 23 °C, 2 h) reached 1341 U/g. Compared with the free form, the immobilized enzyme possesses a slightly higher optimal pH value and a wider pH-activity profile, superior thermal stability, and a higher Km value. Reusability of the immobilized mixed protease indicated that >70% of the original activity was retained after having been recycled six times.

  5. Trypsin-like serine peptidase profiles in the egg, larval, and pupal stages of Aedes albopictus

    PubMed Central

    2013-01-01

    Background Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. Methods Ae. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC. Results The proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases. Conclusion The preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression

  6. Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase.

    PubMed

    Burchett, Gina G; Folsom, Charles G; Lane, Kimberly T

    2015-01-01

    β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

  7. Native Plants, Native Knowledge: Insights from Judy Bluehorse Skelton.

    ERIC Educational Resources Information Center

    Reed, Bracken

    2003-01-01

    Judy Bluehorse Skelton is an herbalist of Native American descent who conducts field trips to identify plants and classroom activities to demonstrate their uses. She also works with Portland (Oregon) schools developing culturally appropriate strategies for presenting Native American content. She encourages students to look at events such as the…

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

    SciTech Connect

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

    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.

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

    SciTech Connect

    Singer, P.T.; Berman, L.E.; Cai, Z.; Mangel, W.F.; Jones, K.W.; Sweet, R.M. ); Carty, R.P. . Dept. of Biochemistry); Schlichting, I. . 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.

  10. Snake venom toxins. The amino acid sequence of toxin Vi2, a homologue of pancreatic trypsin inhibitor, from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Strydom, D J

    1977-04-25

    The amino acid sequence of venom component Vi2, a protein of low toxicity from Dendroaspis polylepis polylepis venom was determined by automatic sequence analysis in combination with sequence studies on tryptic peptides. This protein, the most retarded fraction of this venom on a cation-exchange resin, is a homologue of bovine pancreatic trypsin inhibitor consisting of a single chain of 57 amino acid residues containing six half-cystine residues. The active site lysyl residue of bovine trypsin inhibitor is conserved in Vi2 although large differences are found in the rest of the molecule.

  11. Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body.

    PubMed

    Xiaopeng, Bai; Tanaka, Yoshimasa; Ihara, Eikichi; Hirano, Katsuya; Nakano, Kayoko; Hirano, Mayumi; Oda, Yoshinao; Nakamura, Kazuhiko

    2017-02-15

    Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK1) and NK2 antagonists, but not by an NK3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK1, NK2 and NK3) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK1 and NK2 antagonists, but not by the NK3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK1/2. Gap junctions were indispensable in this tachykinin-induced response.

  12. An anomalous behavior of trypsin immobilized in alginate network.

    PubMed

    Ganachaud, Chrystelle; Bernin, Diana; Isaksson, Dan; Holmberg, Krister

    2013-05-01

    Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N α-benzoyl-L-arginine-4-nitroanilide hydrochloride (L-BAPNA), and in the presence or absence of D-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of D-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with D-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus L-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.

  13. A bone sample cleaning method using trypsin for the isolation of DNA.

    PubMed

    Li, Richard; Liriano, Lidissy

    2011-11-01

    Cleaning the surface of bone samples is a necessary step to remove contaminants prior to isolating DNA for forensic DNA analysis. In this study, a simple trypsin method for cleaning bone samples prior to DNA isolation was developed. Cleaning the surface of human bone samples was achieved by the application of trypsin solution. Light microscopy and scanning electron microscopy results indicated that trypsin treatment was effective in removing the outer surface of bone samples. The yield of DNA isolated from trypsin-treated bone samples was sufficient for subsequent short tandem repeat (STR) analysis. STR analysis revealed no adverse effect on the DNA profile after the trypsin treatment. The data suggest that this trypsin method can potentially be an alternative cleaning method to mechanical cleaning methods.

  14. Earth's Caretakers: Native American Lessons.

    ERIC Educational Resources Information Center

    Nyberg, Lisa M., Ed.

    Written by Native American teachers and by teachers of Native Americans, this book presents examples of ways to learn respect for the Earth and its people. The hope is that students will learn to walk softly upon the Earth and to respect all living things. Lessons and activities engage elementary and middle school students in a four-step…

  15. Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives.

    PubMed

    Monceau, Karine; Arca, Mariangela; Leprêtre, Lisa; Mougel, Florence; Bonnard, Olivier; Silvain, Jean-François; Maher, Nevile; Arnold, Gérard; Thiéry, Denis

    2013-01-01

    Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.

  16. Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives

    PubMed Central

    Monceau, Karine; Arca, Mariangela; Leprêtre, Lisa; Mougel, Florence; Bonnard, Olivier; Silvain, Jean-François; Maher, Nevile; Arnold, Gérard; Thiéry, Denis

    2013-01-01

    Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective. PMID:23823754

  17. Probing the binding of trypsin to glutathione-stabilized gold nanoparticles in aqueous solution.

    PubMed

    Wang, Gongke; Liu, Xingbing; Yan, Changling; Bai, Guangyue; Lu, Yan

    2015-11-01

    We investigate the interaction of trypsin with glutathione-stabilized Au nanoparticles (NPs) using fluorescence, synchronous fluorescence and ultraviolet (UV) absorption spectroscopy. We find that trypsin binds strongly to the Au NPs with a static quenching mechanism, and that the interaction is characteristic of positive cooperative binding. Furthermore, we determine the binding constants and the thermodynamic parameters, which suggest that the main binding forces between the glutathione-stabilized Au NPs and trypsin are electrostatic interactions and hydrogen bonding. Analysis of UV-vis absorption spectra suggests that aggregation of the Au NPs occurs in the trypsin/Au NPs system, which significantly alters the conformation of the protein.

  18. Effect of cigarette smoke on human serum trypsin inhibitory capacity and antitrypsin concentration

    SciTech Connect

    Chowdhury, P.; Bone, R.C.; Louria, D.B.; Rayford, P.L.

    1982-07-01

    Investigation of the effect of cigarette smoke on the serum trypsin inhibitory capacity (TIC) and antitrypsin content in 89 smokers compared with 37 nonsmokers revealed that cigarette smoking is associated with a significantly lower level of TIC. No alteration in serum antitrypsin content was found because of cigarette smoking. Further analysis of the data indicated a correlation between the magnitude of smoking and the reduction in serum TIC. The reduction of TIC in cigarette smokers is consistent with the recent findings of decreased alpha 1-antitrypsin activity in rat lung and the reduced elastase inhibitory capacity per mg of alpha 1-antitrypsin found in the serum of smokers. The decrease in TIC in the serum of smokers, in addition to the reported decrease in elastolytic activity, may be useful in explaining the pathogenesis of emphysema frequently found in smokers.

  19. [Proteolytic enzymes and trypsin inhibitors of higher plants under stress conditions].

    PubMed

    Domash, V I; Sharpio, T P; Zabreĭko, S A; Sosnovskaia, T F

    2008-01-01

    The response of the components of a protease-inhibitor system of legume and cereal crops to stress factors was studied. It was found that salinization, heavy metal ions, and phytopathogenic flora inhibit the activity of neutral, acidic, and alkaline proteases at early stages of seed germination, the degree of the inhibition of the endoprotease activity being dependent on the index of tolerance of legume and cereal crops. It was shown that, in response to unfavorable conditions, accumulation of trypsin inhibitors occurs, which is accompanied by the appearance of new protein components, as indicated by electrophoresis. The results confirm the presumption that serine protease inhibitors are involved in the response of plants to stress factors.

  20. Study of secondary specificity of enteropeptidase in comparison with trypsin.

    PubMed

    Mikhailova, A G; Likhareva, V V; Vaskovsky, B V; Garanin, S K; Onoprienko, L V; Prudchenko, I A; Chikin, L D; Rumsh, L D

    2004-08-01

    A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-(downward arrow)-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM(-1) x min(-1)) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM(-1) x min(-1)).

  1. Functional analysis of five trypsin-like protease genes in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

    PubMed

    Li, Ya-Li; Hou, Ming-Zhe; Shen, Guang-Mao; Lu, Xue-Ping; Wang, Zhe; Jia, Fu-Xian; Wang, Jin-Jun; Dou, Wei

    2017-03-01

    Insect midgut proteases catalyze the release of free amino acids from dietary proteins and are essential for insect normal development. To date, digestive proteases as potential candidates have made great progress in pest control. To clarify the function of trypsin-like protease genes in the digestive system of Bactrocera dorsalis, a serious pest of a wide range of tropical and subtropical fruit and vegetable crops, five trypsin genes (BdTry1, BdTry2, BdTry3, BdTry4 and BdTry5) were identified from transcriptome dataset, and the effects of feeding condition on their expression levels were examined subsequently. RNA interference (RNAi) was applied to further explore their function on the growth of B. dorsalis. The results showed that all the BdTrys in starving midgut expressed at a minimal level but up-regulated upon feeding (except BdTry3). Besides, RNAi by feeding dsRNAs to larvae proved to be an effective method to cause gene silencing and the mixed dsRNAs of the five BdTrys slowed larvae growth of B. dorsalis. The current data suggest that trypsin genes are actively involved in digestion process of B. dorsalis larvae and thereafter play crucial roles in their development.

  2. Human C3 and C5: subunit structure and modifications by trypsin and C42-C423.

    PubMed

    Nilsson, U R; Mandle, R J; McConnell-Mapes, J A

    1975-02-01

    The subunit composition of human C3 and C5 was analyzed. Acrylamide gel electrophoresis of the fully reduced and dissociated proteins disclosed a similar structure, consisting of one alpha and beta subunit, linked together by one or more disulfide bonds. The approximate molecular weights for the alpha and beta subunits of C3 as well as C5 were 140,000 and 80,000 respectively. C42 caused cleavage solely of C3alpha, whereas trypsin affected both C3alpha as well as C3beta. A characteristic subunit modification by both enzmes indicated that C3alpha constitutes the source of C3a. C423 as well as trypsin exclusively affect C5alpha. C5a therefore appears to originate from the C5alpha subunit. The mode of primary cleavage by C423 and trypsin differs, giving rise to different forms of C5b. The questions is raised if multiple forms of C5a also exist. It appeared from our studies that certain forms of C5b may retain portions of the alpha subunit, which could potentially release some biologically active split products following secondary cleavage by the appropriate enzyme.

  3. Effects of Concentration and Reaction Time of Trypsin, Pepsin, and Chymotrypsin on the Hydrolysis Efficiency of Porcine Placenta

    PubMed Central

    Jung, Kyung-Hun; Choi, Ye-Chul; Chun, Ji-Yeon; Min, Sang-Gi

    2014-01-01

    This study investigated the effects of three proteases (trypsin, pepsin and chymotrypsin) on the hydrolysis efficiency of porcine placenta and the molecular weight (Mw) distributions of the placental hydrolysates. Because placenta was made up of insoluble collagen, the placenta was gelatinized by applying thermal treatment at 90 ℃ for 1 h and used as the sample. The placental hydrolyzing activities of the enzymes at varying concentrations and incubation times were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GPC). Based on the SDS-PAGE, the best placental hydrolysis efficiency was observed in trypsin treatments where all peptide bands disappeared after 1 h of incubation as compared to 6 h of chymotrypsin. Pepsin hardly hydrolyzed the placenta as compared to the other two enzymes. The Mw distribution revealed that the trypsin produced placental peptides with Mw of 106 and 500 Da. Peptides produced by chymotrypsin exhibited broad ranges of Mw distribution (1-20 kDa), while the pepsin treatment showed Mw greater than 7 kDa. For comparisons of pre-treatments, the subcritical water processing (37.5 MPa and 200 ℃ of raw placenta improved the efficiency of tryptic digestions to a greater level than that of a preheating treatment (90 ℃ for 1 h). Consequently, subcritical water processing followed by enzymatic digestions has the potential of an advanced collagen hydrolysis technique. PMID:26760932

  4. Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins.

    PubMed

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B; Brinch-Pedersen, Henrik

    2014-01-01

    Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni(2+)-affinity chromatography. Purified protein was evaluated by SDS-PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km=12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60°C, thermal stability T50 value was 44°C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS-PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.

  5. Trypsin from the processing waste of the lane snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents.

    PubMed

    Espósito, Talita S; Marcuschi, Marina; Amaral, Ian P G; Carvalho, Luiz B; Bezerra, Ranilson S

    2010-05-26

    A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

  6. Enculturation, Perceived Stress, and Physical Activity: Implications for Metabolic Risk among the Yup’ik – The Center for Alaska Native Health Research Study

    PubMed Central

    Bersamin, Andrea; Wolsko, Christopher; Luick, Bret; Boyer, Bert; Lardon, Cecile; Hopkins, Scarlett; Stern, Judith S.; Zidenberg-Cherr, Sheri

    2013-01-01

    Objectives American Indians and Alaska Natives report among the lowest levels of physical activity in the U.S, but there is very little systematic research examining the determinants of physical activity patterns in these populations. This study investigated the relationships between enculturation (or, cultural traditionality), psychosocial stress, and physical activity in a community-based sample of Yup’ik women and men living in rural Alaska Native communities. Associations between these variables and several metabolic risk factors were also examined. Design A sample of 488 Yup’ik participants (284 women and 204 men) from 6 villages in the Yukon-Kuskokwim Delta region completed a wellness survey and an array of physiological assessments (e.g., BMI, blood pressure). A subset of 179 participants also completed a 3-day pedometer assessment of physical activity. Results Multivariate linear regression models indicated that participants who were more enculturated (i.e. living more of a traditional lifestyle) and who experienced lower levels of psychosocial stress were significantly more physically active. In turn, lower levels of psychosocial stress and higher levels of physical activity were both associated with lower BMI, lower percent body fat, and lower waist circumference. Conclusions Findings underscore the importance of gaining a culturally-specific understanding of physical activity patterns in indigenous groups in order to inform effective health promotion strategies. PMID:23297688

  7. Native American Folklorist: Paul Goble.

    ERIC Educational Resources Information Center

    Brodie, Carolyn S.

    1998-01-01

    Presents ideas for extending activities focusing on Paul Goble's books and Native Americans. Provides an annotated bibliography of Goble's books and lists audio recordings, video recordings, and biographical information. (PEN)

  8. Differences in Overweight and Obesity among Children from Migrant and Native Origin: The Role of Physical Activity, Dietary Intake, and Sleep Duration.

    PubMed

    Labree, Wim; van de Mheen, Dike; Rutten, Frans; Rodenburg, Gerda; Koopmans, Gerrit; Foets, Marleen

    2015-01-01

    A cross-sectional survey was performed to examine to what degree differences in overweight and obesity between native Dutch and migrant primary school children could be explained by differences in physical activity, dietary intake, and sleep duration among these children. Subjects (n=1943) were primary school children around the age of 8-9 years old and their primary caregivers: native Dutch children (n=1546), Turkish children (n=93), Moroccan children (n=66), other non-western children (n=105), and other western children (n=133). Multivariate regressions and logistic regressions were used to examine the relationship between migrant status, child's behavior, and BMI or prevalence of overweight, including obesity (logistic). Main explanatory variables were physical activity, dietary intake, and sleep duration. We controlled for age, sex, parental educational level, and parental BMI. Although sleep duration, dietary intake of fruit, and dietary intake of energy-dense snacks were associated with BMI, ethnic differences in sleep duration and dietary intake did not have a large impact on ethnic differences in overweight and obesity among children from migrant and native origin. It is suggested that future preventive strategies to reduce overweight and obesity, in general, consider the role of sleep duration. Also, cross-cultural variation in preparation of food among specific migrant groups, focusing on fat, sugar, and salt, deserves more attention. In order to examine which other variables may clarify ethnic differences in overweight and obesity, future research is needed.

  9. Occurrence of trypsin-like protease in cardamom (Elettaria cardamomum Maton).

    PubMed

    Josephrajkumar, A; Chakrabarty, Romit; Thomas, George

    2005-08-01

    Occurrence of trypsin-like protease in fresh cardamom (Elettaria cardamomum Maton) seeds, as evidenced by the benzoyl-arg-p-nitroanilide (BApNA) hydrolyzing ability of the seed enzyme preparation under alkaline condition is reported for the first time. The enzyme has a pH and temperature optima as 8 and 45 degrees C, respectively. It is inhibited by aprotinin and phenylmethyl sulfonyl fluoride (PMSF) in a dose-dependent manner, suggesting the presence of serine residues at the active site. The enzyme had a V(max) of 98.01 nmoles p-nitroaniline released per min per mg protein and K(m) of 0.0684 mM with BApNA as substrate. Addition of aprotinin (75.75 microM) increased K(m) value by three-folds, whereas the V(max) was reduced by 23%.

  10. Trypsin isozymes in the lobster Panulirus argus (Latreille, 1804): from molecules to physiology.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Perdomo-Morales, Rolando; Montero-Alejo, Vivian; Moyano, Francisco Javier; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

    2015-01-01

    Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology.

  11. Removal of dentin matrix proteoglycans by trypsin digestion and its effect on dentin bonding.

    PubMed

    Bedran-Russo, Ana Karina B; Pereira, Patricia N R; Duarte, Wagner R; Okuyama, Katsushi; Yamauchi, Mitsuo

    2008-04-01

    The aim of this study was to investigate the effect of trypsin digestion on removal of chondroitin sulfate proteoglycans (CS-PGs) in demineralized dentin, and subsequent dentin bonding. Bovine dentin fragments were demineralized, treated with or without trypsin, stained with cupromeronic blue, and observed under transmission electron microscopy. Demineralized sections with or without trypsin digestion were also subjected to immunohistochemical analysis with anti-chondroitin-4-sulfate (C4S) monoclonal antibody, 2-B-6. The presence of galactosamine and glucosamine in the trypsin digest was confirmed by amino acid analysis. Bond strength testing was performed on trypsin treated and control specimens where samples were either kept moist or dried and re-wet, then bonded. Bond strength significantly decreased after trypsin treatment (p < 0.05). TEM, immunohistochemical, and amino acid analyses demonstrated that trypsin digestion efficiently removed C4S-PGs from demineralized dentin matrix. This study indicates that the detrimental effects observed on dentin bonding by trypsinization may be due in part to the removal/cleavage of the C4S-PGs, and further underscore the importance of C4S-PGs on dentin bonding.

  12. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of...

  13. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of...

  14. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of...

  15. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of...

  16. Isolation and characterization of a novel trypsin inhibitor from fresh lily bulbs.

    PubMed

    Zhang, Xiaoqing; Wang, Hexiang; Ng, Tzi Bun

    2008-04-01

    A 17-kDa trypsin inhibitor was isolated from fresh lily bulbs with an isolation procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. Its N-terminal sequence displayed similarity to a short segment of the sequences of the Populus tremula trypsin inhibitor, a putative trypsin inhibitor from Arabidopsis thaliana and sporamin B from sweet potato. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC (50) value of 1.3 microM. There was a stimulatory effect on macrophage production of nitric oxide. Unlike field bean trypsin inhibitor it did not inhibit [methyl-(3)H]thymidine incorporation by leukemia L1210 cells and MBL2 cells when tested up to 100 microM. In contrast to broad bean trypsin inhibitor, there was no inhibitory effect on HIV-1 reverse transcriptase when lily bulb trypsin inhibitor was tested up to 100 microM. The present report is one of the very few on bulbs in contrast to the voluminous literature on seeds.

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

    PubMed Central

    Sai, Wenbo; Tian, Hong; Yang, Kangmin; Tang, Daoqi; Bao, Jinxiao; Ge, Yang; Song, Xiaoda; Zhang, Yu; Luo, Cheng; Gao, Xiangdong; Yao, Wenbing

    2017-01-01

    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. PMID:28282854

  18. Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics

    SciTech Connect

    Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.

    2011-07-01

    Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

  19. [Chymotrypsin and trypsin inhibitor isolated from potato tubers].

    PubMed

    Revina, T A; Parfenov, I A; Gvozdeva, E L; Gerasimova, N G; Valueva, T A

    2011-01-01

    Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., Zhukov's Jubilee breed) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-Sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for Carlsberg subtilisin. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.

  20. Precursor of kunitz trypsin inhibitor in soybean seeds

    SciTech Connect

    McGrain, A.; Chen, J.; Tan-Wilson, A. )

    1990-05-01

    Kunitz soybean trypsin inhibitor (KSTI) appears to be synthesized in precursor form which is converted by proteolytic digestion to the mature form of KSTI. Two forms of anti-cross-reacting material are evident when Western blots of extracts of developing seeds are analyzed. The precursor form increases to maximum levels as seed lengths increase to 11 mm. As the seed matures to 13 mm and turns yellow, precursor levels decrease while mature KSTI levels increase. The conversion of precursor to mature form could be demonstrated in vitro in seed extracts. The conversion could also be demonstrated in excised seeds pulse-labeled with ({sup 14}C)-leucine as loss of radioactivity from the precursor and appearance in the mature KSTI form.

  1. Ultrasound speed and attenuation in progressive trypsin digested articular cartilage.

    PubMed

    Niu, HaiJun; Li, LiFeng; Sun, Feng; Yan, Yan; Wang, YueXiang; Li, DeYu; Fan, YuBo

    2011-11-01

    Subtle changes of articular cartilage (AC) can lead to tissue degeneration and even osteoarthritis (OA). The early degeneration of AC is closely related to a change in proteoglycans (PG) content. The observation of PG is therefore an appropriate way of studying OA and evaluating the degree of AC degeneration. In this study, 20 cartilage-bone samples were prepared from normal porcine femoral condyle cartilage and 10 samples were digested over 2 h using 0.25% trypsin solution. The dynamic process of PG-digestion was explored using a conventional A-mode ultrasound (US) experimental system with a 10 MHz center frequency. Quantitative acoustic parameters were calculated from ultrasonic radio-frequency echo signals and included US speed (USS), US amplitude attenuation coefficient (UAA) and broadband US attenuation coefficient (BUA). The experimental results showed that the conventional A-mode ultrasound is valuable for tracking the degree of PG-digestion. Histology also confirmed the validity of the ultrasound observations. For every AC sample, the degree of PG-digestion within a given time was different and was affected by individual differences. After two hours of degeneration, USS showed a mean decrease of 0.4% (P<0.05). UAA was significantly lower after a two-hour PG depletion period (from (2.45±0.23) to (2.28±0.41) dB mm⁻¹). BUA showed no significant differences during this process. In conclusion, conventional ultrasound can provide useful information about trypsin-induced progressive PG depletion in AC and can reflect variations of PG content via the quantitative acoustic parameters USS and UAA. The results of this study may be used to identify an indirect indicator of cartilage matrix integrity and OA disease progression.

  2. Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated.

    PubMed

    Kludkiewicz, Barbara; Kodrík, Dalibor; Grzelak, Krystyna; Nirmala, Xavier; Sehnal, Frantisek

    2005-10-01

    Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited alpha-chymotrypsin and the non-serine proteinases.

  3. Relationship between Single Nucleotide Polymorphisms in the Peroxisome Proliferator-Activated Receptor Gamma Gene and Fatty Acid Composition in Korean Native Cattle

    PubMed Central

    Lee, Jea-young; Ha, Jae-jung; Park, Yong-soo; Yi, Jun-koo; Lee, Seunguk; Mun, Seyoung; Han, Kyudong; Kim, J.-J.; Kim, Hyun-Ji; Oh, Dong-yep

    2016-01-01

    The peroxisome proliferator-activated receptor gamma (PPARγ) gene plays an important role in the biosynthesis process controlled by a number of fatty acid transcription factors. This study investigates the relationships between 130 single-nucleotide polymorphisms (SNPs) in the PPARγ gene and the fatty acid composition of muscle fat in the commercial population of Korean native cattle. We identified 38 SNPs and verified relationships between 3 SNPs (g.1159-71208 A>G, g.42555-29812 G>A, and g.72362 G>T) and the fatty acid composition of commercial Korean native cattle (n = 513). Cattle with the AA genotype of g.1159-71208 A>G and the GG genotype of g.42555-29812 G>A and g.72362 G>T had higher levels of monounsaturated fatty acids and carcass traits (p<0.05). The results revealed that the 3 identified SNPs in the PPARγ gene affected fatty acid composition and carcass traits, suggesting that these 3 SNPs may improve the flavor and quality of beef in commercial Korean native cattle. PMID:26732443

  4. Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells.

    PubMed

    Malysz, John; Rovner, Eric S; Petkov, Georgi V

    2013-07-01

    Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

  5. Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

    PubMed

    Kowalska, Jolanta; Pszczoła, Katarzyna; Wilimowska-Pelc, Anna; Lorenc-Kubis, Irena; Zuziak, Ewa; Ługowski, Mateusz; Łegowska, Anna; Kwiatkowska, Anna; Sleszyńska, Małgorzata; Lesner, Adam; Walewska, Aleksandra; Zabłotna, Ewa; Rolka, Krzysztof; Wilusz, Tadeusz

    2007-06-01

    Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.

  6. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency.

    PubMed

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-03-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

  7. Native Americans with Diabetes

    MedlinePlus

    ... Read the MMWR Science Clips Native Americans with Diabetes Better diabetes care can decrease kidney failure Language: ... between 1996 and 2013. Problem Kidney failure from diabetes was highest among Native Americans. Native Americans are ...

  8. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  9. Depth and orientational dependencies of MRI T(2) and T(1ρ) sensitivities towards trypsin degradation and Gd-DTPA(2-) presence in articular cartilage at microscopic resolution.

    PubMed

    Wang, Nian; Xia, Yang

    2012-04-01

    Depth and orientational dependencies of microscopic magnetic resonance imaging (MRI) T(2) and T(1ρ) sensitivities were studied in native and trypsin-degraded articular cartilage before and after being soaked in 1 mM Gd-DTPA(2-) solution. When the cartilage surface was perpendicular to B(0), a typical laminar appearance was visible in T(2)-weighted images but not in T(1ρ)-weighted images, especially when the spin-lock field was high (2 kHz). At the magic angle (55°) orientation, neither T(2)- nor T(1ρ)-weighted image had a laminar appearance. Trypsin degradation caused a depth- and orientational-dependent T(2) increase (4%-64%) and a more uniform T(1ρ) increase at a sufficiently high spin-lock field (55%-81%). The presence of the Gd ions caused both T(2) and T(1ρ) to decrease significantly in the degraded tissue (6%-38% and 44%-49%, respectively) but less notably in the native tissue (5%-10% and 16%-28%, respectively). A quantity Sensitivity was introduced that combined both the percentage change and the absolute change in the relaxation analysis. An MRI experimental protocol based on two T(1ρ) measurements (without and with the presence of the Gd ions) was proposed to be a new imaging marker for cartilage degradation.

  10. On the Effect of Native SiO2 on Si over the SPR-mediated Photocatalytic Activities of Au and Ag Nanoparticles.

    PubMed

    Wang, Jiale; de Freitas, Isabel; Alves, Tiago; Ando, Romulo A; Fang, Zebo; Camargo, Pedro

    2017-04-11

    In hybrid materials containing plasmonic nanoparticles such as Au and Ag, charge transfer processes from and to Au or Ag can affect both activities and selectivity in plasmonic catalysis. Inspired by the widespread utilization of commercial Si wafers in SERS studies, we investigated herein the effect of the native SiO2 layer on Si wafers over the SPR-mediated activities of the Au and Ag NPs. We prepared SERS-active plasmonic comprised of Au and Ag NPs deposited onto a Si wafer. Here, two kinds of Si wafers were employed: Si having a native oxide surface layer (Si/SiO2) and Si without a native oxide surface layer (Si). This led to Si/SiO2/Au, Si/SiO2/Ag, Si/Au, and Si/Ag NPs. The SPR-mediated oxidation of p-aminothiophenol (PATP) to p,p'-dimercaptoazobenzene (DMAB) was employed as a model transformation. By comparing the performances and band structures for the Si/Au and Si/Ag relative to Si/SiO2/Au and Si/SiO2/Ag NPs, it was found that the presence of a SiO2 layer was crucial to enable higher SPR-mediated PATP to DMAB conversions. The SiO2 layer acts preventing the charge transfer of SPR-excited hot electrons from Au or Ag nanoparticles to the Si substrate. This enabled SPR-excited hot electrons to be transferred to adsorbed O2 molecules, which then participate in the selective oxidation of PATP to DMAB. In the absence of a SiO2 layer, SPR-excited hot electrons are preferentially transferred to Si instead of adsorbed O2 molecules, leading to much lower PATP oxidation.

  11. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    SciTech Connect

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

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

    PubMed Central

    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. PMID:24523715

  13. Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

    PubMed

    Hu, YuanYuan; Qian, Junqing; Guo, Hui; Jiang, ShengLan; Zhang, Zheng

    2015-07-01

    Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

  14. Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin.

    PubMed

    Rivera-Burgos, Dinelia; Regnier, Fred E

    2013-02-01

    Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin-IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin- IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.

  15. Digestive Proteolytic Activity in the Sunn Pest, Eurygaster integriceps

    PubMed Central

    Hosseininaveh, Vahid; Bandani, Alireza; Hosseininaveh, Fatemeh

    2009-01-01

    The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolytic activity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolytic activity was measured at different pH. Maximum proteolytic activity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolytic activity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolytic activity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolytic activity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis uisng native-PAGE. Kinetic studies showed a km value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolytic activity of the trypsin

  16. Trypsin-specific Inhibitors from the Macrolepiota procera, Armillaria mellea and Amanita phalloides wild mushrooms.

    PubMed

    Lukanc, Tjaša; Brzin, Jože; Kos, Janko; Sabotič, Jerica

    2017-01-01

    Wild growing mushrooms are a rich source of novel proteins with unique features. We have isolated and characterized trypsin inhibitors from two edible mushrooms, the honey fungus (Armillaria mellea) and the parasol mushroom (Macrolepiota procera), and from the poisonous death cap (Amanita phalloides). The trypsin inhibitors isolated: armespin, macrospin and amphaspin, have similar molecular masses, acidic isoelectric points and are not N-glycosylated. They are very strong trypsin inhibitors and weak chymotrypsin inhibitors. They are resistant to exposure to high temperatures and withstand extreme pH values. These exceptional characteristics are advantageous for their potential use in biotechnology, agriculture and medicine.

  17. Resolution of two native monomeric 90kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes.

    PubMed

    Simpson, Philippa J L; McKinzie, Audra A; Codd, Rachel

    2010-07-16

    The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

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

    PubMed

    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.

  19. Advocacy for Native American Indian and Alaska Native Clients and Counselees.

    ERIC Educational Resources Information Center

    Herring, Roger

    Helping professionals need to be more informed and more active advocates for proactive counseling strategies with Native American Indian and Alaska Native peoples. The paper discusses the major advocacy needs of these populations. The negative impact of historical and contemporary discriminatory policies and practices on Native peoples has…

  20. Native Aging Visions: A Resource for Native Elders. Volume 1, 1994-97.

    ERIC Educational Resources Information Center

    Native Aging Visions, 1997

    1997-01-01

    This volume of newsletters reports on the activities and research projects of the National Resource Center on Native American Aging located at the University of North Dakota, Grand Forks. The Center studies health issues and access problems facing American Indian, Alaska Native, and Native Hawaiian elders. Specifically, the resource center was…

  1. Functional divergence in tandemly duplicated Arabidopsis thaliana trypsin inhibitor genes.

    PubMed Central

    Clauss, M J; Mitchell-Olds, T

    2004-01-01

    In multigene families, variation among loci and alleles can contribute to trait evolution. We explored patterns of functional and genetic variation in six duplicated Arabidopsis thaliana trypsin inhibitor (ATTI) loci. We demonstrate significant variation in constitutive and herbivore-induced transcription among ATTI loci that show, on average, 65% sequence divergence. Significant variation in ATTI expression was also found between two molecularly defined haplotype classes. Population genetic analyses for 17 accessions of A. thaliana showed that six ATTI loci arranged in tandem within 10 kb varied 10-fold in nucleotide diversity, from 0.0009 to 0.0110, and identified a minimum of six recombination events throughout the tandem array. We observed a significant peak in nucleotide and indel polymorphism spanning ATTI loci in the interior of the array, due primarily to divergence between the two haplotype classes. Significant deviation from the neutral equilibrium model for individual genes was interpreted within the context of intergene linkage disequilibrium and correlated patterns of functional differentiation. In contrast to the outcrosser Arabidopsis lyrata for which recombination is observed even within ATTI loci, our data suggest that response to selection was slowed in the inbreeding, annual A. thaliana because of interference among functionally divergent ATTI loci. PMID:15082560

  2. Native American Discursive Tactic

    ERIC Educational Resources Information Center

    Black, Jason Edward

    2013-01-01

    This essay derives from a course called ‘"The Rhetoric of Native America,’" which is a historical-critical survey of Native American primary texts. The course examines the rhetoric employed by Natives to enact social change and to build community in the face of exigencies. The main goal of exploring a native text (particularly, Simon…

  3. Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin

    PubMed Central

    Tomar, Sakshi; Patil, Dipak N.; Datta, Manali; Tapas, Satya; Preeti; Chaudhary, Anshul; Sharma, Ashwani K.; Tomar, Shailly; Kumar, Pravindra

    2009-01-01

    The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 Å resolution and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 57.1, c = 120.1 Å. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor–trypsin complex per asymmetric unit, with a solvent content of 45%. PMID:19923745

  4. Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans.

    PubMed

    Wilson, K A; Tan-Wilson, A L

    1983-01-01

    The mung bean (Vigna radiata (L.) Wilczek) trypsin inhibitor (MBTI) is rapidly modified by limited proteolysis during the early stages of seedling growth. Using an electrophoretic assay that separates the unmodified inhibitor (MBTI-F) and the first two modified species (MBTI-E and -C), a pH optimum of approximately 4 was found for the modification reaction. The inhibitor modifying activity is initially low in ungerminated seeds, with the reaction F leads to E being the primary reaction catalyzed. Activity catalyzing the production of MBTI-C appears on the first day of germination. This activity (F leads to E leads to C) increases up to 6 days after inhibition, at which time the cotyledons begin to abscise. The activity converting MBTI-F and -E to MBTI-C was strongly inhibited by phenylmethylsulfonyl fluoride (3.3 mM) but only weakly by iodoacetate (9 mM) and not at all by pepstatin A (9 microM), leupeptin (18 microM), or EDTA (5 mM). These results suggest the involvement of proteinases other than the major endopeptidase of the germinating seed, vicilin peptidohydrolase. This conclusion is further supported by gel filtration of the extracts of cotyledons on Sephacryl S-200. At least three proteinases are present in germinated cotyledons capable of modifying MBTI-F to MBTI-C and/or -E. All are distinguishable from vicilin peptidohydrolase on the basis of their molecular weight and inhibition by low molecular weight organic reagents.

  5. The random-coil 'C' fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors.

    PubMed Central

    Haarmann, Claudia S; Green, Daniel; Casarotto, Marco G; Laver, Derek R; Dulhunty, Angela F

    2003-01-01

    The actions of peptide C, corresponding to (724)Glu-Pro(760) of the II-III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM-10 microM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 microM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation-contraction coupling or interactions between the proteins at rest. PMID:12620094

  6. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial infection and inflammation. (b) Classification. Class I (general controls). The device is exempt from...

  7. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial infection and inflammation. (b) Classification. Class I (general controls). The device is exempt from...

  8. Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

    PubMed Central

    Bohe, M; Borgström, A; Lindström, C; Ohlsson, K

    1986-01-01

    Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material. Images PMID:3525612

  9. Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly).

    PubMed

    Gomes, Carlos E M; Barbosa, Aulus E A D; Macedo, Leonardo L P; Pitanga, Joelma C M; Moura, Fabiano T; Oliveira, Adeliana S; Moura, Raniere M; Queiroz, Alexandre F S; Macedo, Francisco P; Andrade, Lúcia B S; Vidal, Márcia S; Sales, Mauricio P

    2005-12-01

    A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.

  10. Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin

    PubMed Central

    Jin, Lei; Roehrl, Michael H. A.; Xiao, Li; He, Xiuyun; Li, Haibin; Ge, Linhu; Shi, Bingyi

    2012-01-01

    Calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20–347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-­transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Å resolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing. PMID:22691791

  11. Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

    PubMed Central

    Vodkin, Lila O.

    1981-01-01

    The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA. Images PMID:16661996

  12. Self-powered sensor for naked-eye detection of serum trypsin.

    PubMed

    Zaccheo, Brian A; Crooks, Richard M

    2011-02-15

    Here, we report a device for the detection of the proteolytic enzyme trypsin, which is a biomarker for pancreatitis. The sensor is self-powered, easy to use, and signals the presence of trypsin via a light-emitting diode (LED) that is visible to the unaided eye. Assay time is ∼3 h, and the limit of detection is 0.5 μg/mL, which is within the range required for detection of trypsin at levels signaling acute pancreatitis. The sensing mechanism relies on trypsin digestion of a gelled protein layer. Partial digestion of the protein layer permits hydroxide penetration and subsequent etching of an underlying Al membrane. Degradation of both the protein and Al layers exposes an underlying Mg anode and closes an electrochemical circuit that produces ∼2.2 V. This is sufficient voltage to illuminate the LED. A logarithmic relationship is observed between the time required for LED illumination and trypsin concentration. The device is equally effective for trypsin dissolved in buffer or serum media.

  13. Astronomy in the Native-Oriented Classroom.

    ERIC Educational Resources Information Center

    Smith, Murray R.

    1984-01-01

    Outlines background, materials, operation, and evaluation of four activities for grades six-nine designed to illustrate how curriculum activities can enhance astronomy concepts and native awareness: "Were Native People Aware of Milky Way Galaxy?,""Constellation Cans,""Travels of the Big Dipper," and "How Did the…

  14. The interaction of alpha-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin.

    PubMed Central

    Klausner, Y S; Rigbi, M; Ticho, T; De Jong, P J; Neginsky, E J; Rinott, Y

    1978-01-01

    The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin. Images PLATE 1 PMID:629742

  15. Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling.

    PubMed

    Sattar, Rabia; Ali, Syed Abid; Kamal, Mustafa; Khan, Aftab Ahmed; Abbasi, Atiya

    2004-02-13

    Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.

  16. Isolation and Characterization of the Promoter and Partial Enhancer Region of the Porcine Inter-α-Trypsin Inhibitor Heavy Chain 4 Gene

    PubMed Central

    Harraghy, Niamh; Mitchell, Timothy J.

    2005-01-01

    A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-α-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1β. However, IL-1β was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter. PMID:16275952

  17. Native Geoscience: Pathways to Knowledge

    NASA Astrophysics Data System (ADS)

    Bolman, J. R.; Seielstad, G.

    2006-12-01

    We are living in a definite time of change. Distinct changes are being experienced in our most sacred and natural environments. This is especially true on Native lands. Native people have lived for millennia in distinct and unique ways. The knowledge of balancing the needs of people with the needs of our natural environments is paramount in all tribal societies. This inherent accumulated knowledge has become the foundation on which to build a "blended" contemporary understanding of western science. The Dakota's and Northern California have embraced the critical need of understanding successful tribal strategies to engage educational systems (K-12 and higher education), to bring to prominence the professional development opportunities forged through working with tribal peoples and ensure the continued growth of Native earth and environmental scientists The presentation will highlight: 1) past and present philosophies on building and maintaining Native/Tribal students in earth and environmental sciences; 2) successful educational programs/activities in PreK-Ph.D. systems; 3) current Native leadership development in earth and environmental sciences; and 4) forward thinking for creating proaction collaborations addressing sustainable environmental, educational and social infrastructures for all people. Humboldt State University (HSU) and the University of North Dakota's Northern Great Plains Center for People and the Environment and the Upper Midwest Aerospace Consortium (UMAC) have been recognized nationally for their partnerships with Native communities. Unique collaborations are emerging "bridging" Native people across geographic areas in developing educational/research experiences which integrate the distinctive earth/environmental knowledge of tribal people. The presentation will highlight currently funded projects and initiatives as well as success stories of emerging Native earth system students and scientists.

  18. Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes.

    PubMed

    Saleh, Sohag N; Albert, Anthony P; Large, William A

    2009-11-15

    We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ET(A) or ET(B) receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ET(A) and ET(B) receptor stimulation activate this conductance. Stimulation of both ET(A) and ET(B) receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ET(A) receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nM) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP(3)), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Moreover, exogenous application of diC8-PIP(3) stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ET(A) receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP(3). In contrast, ET(B) receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP(2)) antibodies and high concentrations of wortmannin (20 microM) which

  19. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

    SciTech Connect

    Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; Hockla, Alexandra; Soares, Alexei S.; Radisky, Evette S.

    2015-07-14

    Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.

  20. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

    DOE PAGES

    Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; ...

    2015-07-14

    Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

  1. Effects of tobacco genetically modified to express protease inhibitor bovine spleen trypsin inhibitor on non-target soil organisms.

    PubMed

    O'Callaghan, Maureen; Brownbridge, Michael; Stilwell, Wendy B; Gerard, Emily M; Burgess, Elisabeth P J; Barraclough, Emma I; Christeller, John T

    2007-01-01

    Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

  2. Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin.

    PubMed

    Chater, Peter Ian; Wilcox, Mathew D; Brownlee, Iain A; Pearson, Jeffrey P

    2015-10-20

    Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase.

  3. Preparation of a novel polymer monolith with functional polymer brushes by two-step atom-transfer radical polymerization for trypsin immobilization.

    PubMed

    Li, Nan; Zheng, Wei; Shen, Ying; Qi, Li; Li, Yaping; Qiao, Juan; Wang, Fuyi; Chen, Yi

    2014-12-01

    Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]-co-glycidyl methacrylate} brushes were fabricated via two-step atom-transfer radical polymerization and used as a trypsin-based reactor in a continuous flow system. This is the first time that atom-transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface-initiated atom-transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis-Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.

  4. Keepers of the Earth. Native American Stories and Environmental Activities for Children [and] Keepers of the Earth--Teacher's Guide.

    ERIC Educational Resources Information Center

    Caduto, Michael J.; Bruchac, Joseph

    When children are allowed to experience stories and activities that help them to care for, and take care of other people and the Earth, they develop a conservation ethic. A collection is presented of carefully chosen North American Indian stories and hands-on activities that promote understanding and appreciation of, empathy for, and responsible…

  5. Use of cholinesterase activity as a biomarker of pesticide exposure used on Costa Rican banana plantations in the native tropical fish Astyanax aeneus (Günther, 1860).

    PubMed

    Mena, F; Azzopardi, M; Pfennig, S; Ruepert, C; Tedengren, M; Castillo, L E; Gunnarsson, J S

    2014-01-01

    In Costa Rica, thousands of tones of agricultural pesticides have been used for decades and their use is continuously increasing due to intensive and expanding production of coffee, pineapple, rice, ornamental plants and bananas. The objective of this study was to evaluate whether choline esterase (ChE) activity could be used as a biomarker of exposure to pesticides in the Costa Rican native fish Astyanax aeneus (characidae). Three methods used in order to evaluate the ChE biomarker were as follows: Laboratory studies where A. aeneus was exposed to organophosphate pesticide (ethoprophos); In situ 48 hr exposure assessment using caging experiments with fish exposed upstream and downstream of banana plantations and ChE activity estimation of in fish captured directly at sites with different degrees of pesticide exposure. Results from the laboratory studies showed that ChE activity in both brain and muscle tissue was significantly lower in fish exposed to ethoprophos than in controls. Fish from the caging experiments showed no difference in ChE activity neither in brain nor in muscle tissue between the four tested sites and was attributed to the short duration of the exposure. Asignificant difference in ChE activity was determined in muscle of fish captured from Laguna Madre de Dios compared to fish from Canal Batán. Although our laboratory results revealed that ChE activity in A. aeneus was highly responsive to ethoprophos, results from field experiments were less conclusive and showed that the captured fish showed large variability in ChE activity and that more research is needed before ChE activity can be used as reliable biomarker of pesticide exposure.

  6. Native American Healing Traditions

    ERIC Educational Resources Information Center

    Portman, Tarrell A. A.; Garrett, Michael T.

    2006-01-01

    Indigenous healing practices among Native Americans have been documented in the United States since colonisation. Cultural encapsulation has deterred the acknowledgement of Native American medicinal practices as a precursor to folk medicine and many herbal remedies, which have greatly influenced modern medicine. Understanding Native American…

  7. Alaska Natives & the Land.

    ERIC Educational Resources Information Center

    Arnold, Robert D.; And Others

    Pursuant to the Native land claims within Alaska, this compilation of background data and interpretive materials relevant to a fair resolution of the Alaska Native problem seeks to record data and information on the Native peoples; the land and resources of Alaska and their uses by the people in the past and present; land ownership; and future…

  8. Native American Homeschooling Association.

    ERIC Educational Resources Information Center

    Rozon, Gina

    2000-01-01

    The Native American Home School Association helps Native parents to provide a good education free from the assimilationist tendencies of public school and to transmit Native values and culture. Discusses various home schooling styles, the effectiveness of home schooling in terms of academic achievement and socialization, and the effectiveness of…

  9. Structure-function relationship of a bio-pesticidal trypsin/chymotrypsin inhibitor from winged bean.

    PubMed

    Banerjee, Sayanika; Giri, Ashok P; Gupta, Vidya S; Dutta, Samir Kumar

    2017-03-01

    Protease inhibitors are essential bio-molecules that serve as a model system for the study of protein structure and protease-protease inhibitor interaction. We here report a bi-functional serine protease inhibitor from winged bean (WBCTI) that completely retains its inhibitory property against trypsin and chymotrypsin even after heating at 70°C. Detailed circular dichroism and fluorescence studies at different temperatures, 30-90°C, have been performed to understand the reason behind thermal stability of the protein. On the basis of our results it appears that WBCTI maintains its canonical structure up to 70°C. Above that the heat induced conformational change becomes irreversible which causes aggregation followed by precipitation of the protein. Moreover, the activity and stability of the secondary structure are found to decrease drastically in presence of dithiothreitol indicating that the protein acquires additional stability for the occurrence of two disulfide bonds. In addition to the structural characterization, an important property of WBCTI against the polyphagous pest Helicoverpa armigera has been explored in present study. WBCTI has showed reasonable inhibition of the mid-gut proteases of H. armigera. In artificial feeding trial through addition of WBCTI in diet resulted in significant growth retardation, delayed pupae formation and higher mortality of H. armigera larvae.

  10. Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase.

    PubMed Central

    De Lorenzo, C.; Dal Piaz, F.; Piccoli, R.; Di Maro, A.; Pucci, P.; D'Alessio, G.

    1998-01-01

    Dimeric seminal RNase (BS-RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N-terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N-terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N-terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface. PMID:9865960

  11. Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

    PubMed Central

    Zhang, Xi

    2015-01-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

  12. Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast.

    PubMed

    Ribeiro, Suzanna F F; Silva, Marciele S; Da Cunha, Maura; Carvalho, André O; Dias, Germana B; Rabelo, Guilherme; Mello, Erica O; Santa-Catarina, Claudete; Rodrigues, Rosana; Gomes, Valdirene M

    2012-03-01

    A 6,000 Da peptide, named CaTI, was isolated from Capsicum annuum L. seeds and showed potent inhibitory activity against trypsin and chymotrypsin. The aim of this study was to determine the effect of CaTI on Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Kluyveromyces marxiannus cells. We observed that CaTI inhibited the growth of S. cerevisiae, K. marxiannus as well as C. albicans and induced cellular agglomeration and the release of cytoplasmic content. No effect on growth was observed in C. tropicalis but morphological changes were noted. In the spot assay, different degrees of sensitivity were shown among the strains and concentrations tested. Scanning electron microscopy showed that S. cerevisiae, K. marxiannus and C. albicans, in the presence of CaTI, exhibited morphological alterations, such as the formation of pseudohyphae, cellular aggregates and elongated forms. We also show that CaTI induces the generation of nitric oxide and interferes in a dose-dependent manner with glucose-stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells.

  13. Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme.

    PubMed

    Knap, A K; Pratt, R F

    1991-01-01

    The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence [Knap & Pratt (1989) Proteins Struct. Funct. Genet. 6, 316-323] for the activation of the active-site thiol group by adjacent functional groups. The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site. The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1. It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234). A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated. A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.

  14. Native sulfur, sulfates and sulfides from the active Campi Flegrei volcano (southern Italy): Genetic environments and degassing dynamics revealed by mineralogy and isotope geochemistry

    NASA Astrophysics Data System (ADS)

    Piochi, Monica; Mormone, Angela; Balassone, Giuseppina; Strauss, Harald; Troise, Claudia; De Natale, Giuseppe

    2015-10-01

    We investigated sulfur-bearing minerals from the Campi Flegrei caldera, southern Italy, in relation to the increase of hydrothermal activity phenomena since 2006, aimed at providing insights into the volcanic system dynamics. Mineral encrustations and muds were sampled between 2013 and 2015 at the long-standing degassing crater of the Solfatara tuff cone and its recently restless north-eastern Pisciarelli slope. Deep-seated sulfides were further separated from two drill cores (AGIP's Mofete boreholes: 1500 m and 2695 m depth). The mineral assemblage and texture of sampled encrustations were determined by X-ray diffraction, optical and scanning electron microscopy and X-ray microanalysis by energy dispersive spectrometry. Native sulfur and alunite dominate among the newly formed mineral phases. Other minerals are mostly alunogen, and locally pickeringite, potassium alum, hematite and pyrite. Mereiterite and amarillite sporadically occur. The mud pools are rich in gypsum, potassium alum and pyrite. Quartz and argillic phases, locally with analcime, are dispersed in the outcropping rocks. δ34S values were determined for shallow subsurface native sulfur (- 5.5 to 0.0‰) and alunite (- 1.7 to - 0.2‰), as well as for the deep-seated pyrite (3.3 to 7.4‰ in the depth range:1500-2695 m). δ18O values were measured for shallow native alunite (4.2 to 7.0‰). Pisciarelli alunite was finally analyzed for its 87Sr/86Sr ratio and 143Nd/144Nd ratios (0.707517 ± 6 and 0.512459 ± 6, respectively). Textural and isotopic data constrain the genesis of alunite at the expense of K-feldspars through rock alteration by hydrothermal fluids. We suggest that the caldera is a low-sulfidation system hosting acid-sulfate deposits in its active degassing area. The acid-sulfate environment developed on an argillitic facies that thins outwards and is characteristic for steam-heated and magmatic-steam environments. These environments developed in relation to the fractured settings that

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

    SciTech Connect

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

    2007-12-01

    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.

  16. Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

    PubMed Central

    do Nascimento Campos Ribeiro, Joycellane Alline; Serquiz, Alexandre Coellho; dos Santos Silva, Priscila Fabíola; Barbosa, Patrícia Batista Barra Medeiros; Sampaio, Tarcísio Bruno Montenegro; de Araújo, Raimundo Fernandes; de Oliveira, Adeliana Silva; Machado, Richele Janaina Araújo; Maciel, Bruna Leal Lima; Uchôa, Adriana Ferreira; dos Santos, Elizeu Antunes; de Araújo Morais, Ana Heloneida

    2015-01-01

    OBJECTIVES: Seeds are excellent sources of proteinase inhibitors, some of which may have satietogenic and slimming actions. We evaluated the effect of a trypsin inhibitor from Tamarindus indica L. seeds on weight gain, food consumption and cholecystokinin levels in Wistar rats. METHODS: A trypsin inhibitor from Tamarindus was isolated using ammonium sulfate (30–60%) following precipitation with acetone and was further isolated with Trypsin-Sepharose affinity chromatography. Analyses were conducted to assess the in vivo digestibility, food intake, body weight evolution and cholecystokinin levels in Wistar rats. Histological analyses of organs and biochemical analyses of sera were performed. RESULTS: The trypsin inhibitor from Tamarindus reduced food consumption, thereby reducing weight gain. The in vivo true digestibility was not significantly different between the control and Tamarindus trypsin inhibitor-treated groups. The trypsin inhibitor from Tamarindus did not cause alterations in biochemical parameters or liver, stomach, intestine or pancreas histology. Rats treated with the trypsin inhibitor showed significantly elevated cholecystokinin levels compared with animals receiving casein or water. CONCLUSION: The results indicate that the isolated trypsin inhibitor from Tamarindus reduces weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin. Thus, the potential use of this trypsin inhibitor in obesity prevention and/or treatment should be evaluated. PMID:25789523

  17. Diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus Opuntia humifusa (Cactaceae) from the United States.

    PubMed

    Silva-Hughes, Alice F; Wedge, David E; Cantrell, Charles L; Carvalho, Camila R; Pan, Zhiqiang; Moraes, Rita M; Madoxx, Victor L; Rosa, Luiz H

    2015-06-01

    The endophytic fungal community associated with the native cactus Opuntia humifusa in the United States was investigated and its potential for providing antifungal compounds. A hundred-eight endophytic fungal isolates were obtained and identified by molecular methods into 17 different taxa of the genera Alternaria, Aureobasidium, Biscogniauxia, Cladosporium, Cryptococcus, Curvularia, Diaporthe, Epicoccum, Paraconiothyrium, Pestalotiopsis and Phoma. The most frequent species associated with O. humifusa were Alternaria sp. 3, Aureobasidium pullulans and Diaporthe sp. The fungal community of O. humifusa had a high richness and diversity; additionally, the species richness obtained indicates that the sample effort was enough to recover the diversity pattern obtained. Six extracts of endophytes showed antifungal properties and (1)H NMR analyses of the extracts of Alternaria sp. 5 Ohu 8B2, Alternaria sp. 3 Ohu 30A, Cladosporium funiculosum Ohu 17C1 and Paraconiothyrium sp. Ohu 17A indicated the presence of functional groups associated with unsaturated fatty-acid olefinic protons and fatty acid methylene and methyl protons. GC-FID analysis of these extracts confirmed the presence of a mixture of different fatty acids. The (1)H NMR analyses of Biscogniauxia mediterranea Ohu 19B extracts showed the presence of aromatic compounds. From the extract of B. mediterranea we isolated the compound 5-methylmellein that displayed moderate antifungal activity against the phytopathogenic fungi Phomopsis obscurans. Our results suggest that native medicinal cacti of the United States can live symbiotically with rich and diverse endophytic communities and may be a source of bioactive molecules, including those able to inhibit or control plant disease pathogens.

  18. Antioxidant, Cytotoxic, and Toxic Activities of Propolis from Two Native Bees in Brazil: Scaptotrigona depilis and Melipona quadrifasciata anthidioides

    PubMed Central

    Bonamigo, Thaliny; Campos, Jaqueline Ferreira; Alfredo, Tamaeh Monteiro; Balestieri, José Benedito Perrella; Cardoso, Claudia Andrea Lima; Paredes-Gamero, Edgar Julian; de Picoli Souza, Kely

    2017-01-01

    Propolis is a natural mixture of compounds produced by various bee species, including stingless bees. This compound has been shown to exhibit antioxidant, antiproliferative, and antitumor activities. The present study aimed to determine the chemical constituents as well as the antioxidant, cytotoxic, and toxic activities of ethanol extracts of propolis obtained from the stingless bees Scaptotrigona depilis and Melipona quadrifasciata anthidioides, which are found in Brazil. Phytosterols, terpenes, phenolic compounds, and tocopherol were identified in the ethanol extracts of propolis (EEPs) in different concentrations. The compounds stigmasterol, taraxasterol, vanilic acid, caffeic acid, quercetin, luteolin, and apigenin were found only in EEP-M. The EEPs were able to scavenge the free radicals 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and protected human erythrocytes against lipid peroxidation, with the latter effect being demonstrated by their antihemolytic activity and inhibition of malondialdehyde formation. The EEPs showed cytotoxic activity against erythroleukemic cells and necrosis was the main mechanism of death observed. In addition, the concentrations at which the EEPs were cytotoxic were not toxic against Caenorhabditis elegans. In this context, it is concluded that EEP-S and EEP-M show antioxidant and cytotoxic activities and are promising bioactive mixtures for the control of diseases associated with oxidative stress and tumor cell proliferation. PMID:28377794

  19. Native oligodeoxynucleotides specifically active against human immunodeficiency virus type 1 in vitro: a G-quartet-driven effect?

    PubMed Central

    Tondelli, L; Colonna, F P; Garbesi, A; Zanella, S; Marongiu, M E; Corrias, S; Loi, A G; La Colla, P

    1996-01-01

    Among a series of unmodified phosphodiester (PO)-oligodeoxynucleotides (PO-ODNs) complementary to some of the human immunodeficiency virus type 1 (HIV-1) regulatory genes, several PO-ODN sequences complementary to the vpr gene (PO-ODNs-a-vpr, where a-vpr is the antisense vpr sequence) emerged as potent inhibitors (at concentrations of 0.8 to 3.3 microM) of HIV-1 multiplication in de novo infected MT-4 cells, while they showed no cytotoxicity for uninfected cells at concentrations up to 100 microM. Unlike phosphorothioate counterparts, PO-ODN-a-vpr sequences were not inhibitory to HIV-2 multiplication in de novo infected C8166 cells and neither prevented the fusion between chronically infected and bystander CD4+ cells nor inhibited the activity of the HIV-1 reverse transcriptase in enzyme assays. Moreover, they were not inhibitory to HIV-1 multiplication in chronically infected cells. Delayed addition experiments showed that PO-ODNs-a-vpr inhibit an event in the HIV-1 replication cycle following adsorption to the host cell, but preceding reverse transcription. Structure-activity relationship studies indicated that the antiviral activity of the test PO-ODN-a-vpr sequences is not related to an antisense mechanism but to the presence, within the active sequences, of contiguous guanine residues. Physical characterization of the test PO-ODNs suggested that the active structure is a tetramer stabilized by G quartets (i.e., four G residues connected by eight hydrogen bonds). PMID:8878576

  20. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  1. Screening of white-rot fungi manganese peroxidases: a comparison between the specific activities of the enzyme from different native producers

    PubMed Central

    2012-01-01

    In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes. Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified. Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa. PMID:23190610

  2. Effects of water stress, organic amendment and mycorrhizal inoculation on soil microbial community structure and activity during the establishment of two heavy metal-tolerant native plant species.

    PubMed

    Fernández, D A; Roldán, A; Azcón, R; Caravaca, F; Bååth, E

    2012-05-01

    Our aim was to examine the effect of water stress on plant growth and development of two native plant species (Tetraclinis articulata and Crithmum maritimum) and on microbial community composition and activity in the rhizosphere soil, following the addition of an organic amendment, namely sugar beet residue (SBR), and/or the inoculation with an arbuscular mycorrhizal (AM) fungus, namely Glomus mosseae, in a non-sterile heavy metal-polluted soil. The AM inoculation did not have any significant effect on plant growth of both species. In T. articulata, SBR increased shoot growth, foliar P, total phospholipid fatty acids (PLFA), fungi-related PLFA, AM fungi-related neutral lipid fatty acid, bacterial gram-positive/gram-negative PLFA ratio and the β-glucosidase and dehydrogenase activities. SBR and AM inoculation increased phosphatase activity in T. articulata plants grown under drought conditions. In both plants, there was a synergistic effect between AM inoculation and SBR on mycorrhizal colonisation under drought conditions. In C. maritimum, the increase produced by the SBR on total amounts of PLFA, bacterial gram-positive-related PLFA and bacterial gram-negative-related PLFA was considerably higher under drought conditions. Our results suggest that the effectiveness of the amendment with regard to stimulating microbial communities and plant growth was largely limited by drought, particularly for plant species with a low degree of mycorrhizal colonisation.

  3. Nature Study Tips: Native American Foods.

    ERIC Educational Resources Information Center

    Russell, Helen Ross

    1984-01-01

    Discusses Native American foods, focusing on Native American cultivated crops, methods of cooking, and methods of preserving food. Includes suggestions for 19 classroom activities, including collecting wild plants used as food, gathering/drying and eating various wild plants and plant products (such as acorns and corn), and making a garden. (JN)

  4. Stennis Space Center celebrates Native American culture

    NASA Technical Reports Server (NTRS)

    2009-01-01

    Famie Willis (left), 2009-2010 Choctaw Indian Princess, displays artifacts during Native American Heritage Month activities at Stennis Space Center on Nov. 24. The celebration featured various Native American cultural displays for Stennis employees to view. Shown above are (l to r): Willis, Elaine Couchman of NASA Shared Services Center, John Cecconi of NSSC and Lakeisha Robertson of the Environmental Protection Agency.

  5. Screening Active Compounds from Garcinia Species Native to China Reveals Novel Compounds Targeting the STAT/JAK Signaling Pathway.

    PubMed

    Xu, Linfeng; Lao, Yuanzhi; Zhao, Yanhui; Qin, Jian; Fu, Wenwei; Zhang, Yingjia; Xu, Hongxi

    2015-01-01

    Natural compounds from medicinal plants are important resources for drug development. In a panel of human tumor cells, we screened a library of the natural products from Garcinia species which have anticancer potential to identify new potential therapeutic leads and discovered that caged xanthones were highly effective at suppressing multiple cancer cell lines. Their anticancer activities mainly depended on apoptosis pathways. For compounds in sensitive cancer line, their mechanisms of mode of action were evaluated. 33-Hydroxyepigambogic acid and 35-hydroxyepigambogic acid exhibited about 1 μM IC50 values against JAK2/JAK3 kinases and less than 1 μM IC50 values against NCI-H1650 cell which autocrined IL-6. Thus these two compounds provided a new antitumor molecular scaffold. Our report describes 33-hydroxyepigambogic acid and 35-hydroxyepigambogic acid that inhibited NCI-H1650 cell growth by suppressing constitutive STAT3 activation via direct inhibition of JAK kinase activity.

  6. Synthesis and characterization of a beta-hairpin peptide that represents a 'core module' of bovine pancreatic trypsin inhibitor (BPTI).

    PubMed

    Carulla, N; Woodward, C; Barany, G

    2000-07-11

    A new strategy for the design and construction of peptide fragments that can achieve defined, nativelike secondary structure is presented. The strategy is based upon the hypothesis that 'core elements' of a protein, synthesized in a single polypeptide molecule, will favor nativelike structure, and that by incorporating a cross-link, nativelike core structure will dominate the ensemble as the more extended conformations are excluded. 'Core elements' are the elements of packed secondary structure that contain the slowest exchanging backbone amide protons in the native protein. The 'core elements' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-sheet (residues 18-24 and 29-35) and the small beta-bridge (residues 43-44). To test the design strategy, we synthesized an 'oxidized core module', which contains the antiparallel strands connected by a modified reverse turn (A27 replaced by D), a natural disulfide cross-link at the open end of the hairpin, and N- and C-termini blocking groups. A peptide with identical sequence but lacking the disulfide cross-link at the open end was used as the 'reduced core module' control. The conformational behavior of both peptides was examined using (1)H NMR spectroscopy. Chemical shift dispersion, chemical shift deviation from random coil values, sequential and long-range NOEs, and H/D amide exchange rates were compared for the two peptides. We conclude that the ensemble of oxidized and reduced core module conformations samples both nativelike 4:4 and non-native 3:5 beta-hairpin structure, and that the oxidized module samples nativelike structure for a greater fraction of the time than the reduced module.

  7. Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

    PubMed

    Lin, Shuang; Lin, Zhenxin; Yao, Guoping; Deng, Chunhui; Yang, Pengyuan; Zhang, Xiangmin

    2007-01-01

    In this study, very easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion and firstly applied for proteome analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Magnetic microspheres with small size were synthesized and modified by 3-glycidoxypropyltrimethoxysilane (GLYMO). Trypsin was immobilized onto magnetic microspheres through only a one-step reaction of its amine group with GLYMO. When these easily prepared trypsin-immobilized magnetic microspheres were applied in microwave-assisted protein digestion, the magnetic microspheres not only functionalized as substrate for trypsin immobilization, but also as an excellent microwave absorber and thus improved the efficiency of microwave-assisted digestion greatly. Cytochrome c was used as a model protein to verify its digestion efficiency. Without any additives such as organic solvents or urea, peptide fragments produced in 15 s could be confidently identified by MALDI-TOF-MS and better digestion efficiency was obtained comparing to conventional in-solution digestion (12 h). Besides, with an external magnet, trypsin could be used repeatedly and at the same time no contaminants were introduced into the sample solution. It was verified that the enzyme maintained high activity after seven runs. Furthermore, reversed-phase liquid chromatography (RPLC) fractions of rat liver extract were also successfully processed using this novel method. These results indicated that this fast and efficient digestion method, which combined the advantages of immobilized trypsin and microwave-assisted protein digestion, will greatly hasten the application of top-down proteomic techniques for large-scale analysis in biological and clinical research.

  8. Central effects of native urotensin II on motor activity, ventilatory movements, and heart rate in the trout Oncorhynchus mykiss.

    PubMed

    Lancien, Frédéric; Leprince, Jérôme; Mimassi, Nagi; Mabin, Dominique; Vaudry, Hubert; Le Mével, Jean-Claude

    2004-10-15

    Urotensin II (UII) has been originally isolated from fish urophysis. However, in fish as in mammals, UII is also produced in brain neurons. Although UII binding sites are widely distributed in the fish central nervous system (CNS), little is known regarding its central activities. In the present study, we have investigated the effects of intracerebroventricular (ICV) administration of synthetic trout UII on the duration of motor activity (ACT; evidenced by bursts of activity on the trace of the ventilatory signal), ventilatory frequency (VF), ventilatory amplitude (VA), and heart rate (HR) in unanesthesized trout, Oncorhynchus mykiss. ICV injection of very low doses of UII (1 and 5 pmol) produced a dose-dependent increase of ACT without affecting VF, VA, or HR. At a higher dose (50 pmol), UII stimulated ACT as well as VF, VA, and HR. ICV injection of trout angiotensin II (5 pmol) did not affect ACT, VF, and VA, but provoked a robust increase in HR. These data provide the first evidence that central administration of UII stimulates motor activity in a nonmammalian vertebrate.

  9. Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.

    PubMed

    Oliveira, Adeliana S; Migliolo, Ludovico; Aquino, Rodrigo O; Ribeiro, Jannison K C; Macedo, Leonardo L P; Andrade, Lucia B S; Bemquerer, Marcelo P; Santos, Elizeu A; Kiyota, Sumika; de Sales, Maurício P

    2007-01-01

    A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.

  10. Inhibition of Amyloid-like Fibril Formation of Trypsin by Red Wines.

    PubMed

    Kotormán, Márta; Kasi, Phanindra Babu; Halász, László; Borics, Attila

    2017-02-14

    The aim of the present study was to examine the potential role and applicability of dietary supplements in reducing the risk of development of amyloid diseases associated with the gastrointestinal tract, such as type II diabetes. Trypsin, a well-known serine protease was used as a model protein in our experiments. The effect of various red wines on the formation of amyloid-like fibrils of trypsin was studied in vitro, in aqueous ethanol, at pH 7.0. Turbidity measurements, aggregation kinetics experiments, Congo red binding assays and electronic circular dichroism spectroscopic measurements were used to follow the aggregation process in the presence or absence of various red wines. The results suggest that red wines effectively inhibit the formation of amyloid-like fibrils of trypsin and the inhibitory effect is dose-dependent. The extent of inhibition was found to be proportional to the total concentration of phenolic compounds.

  11. Increased (/sup 125/I)trypsin-binding in serum from cystic fibrosis patients

    SciTech Connect

    Cox, K.L.; Frates, R.C. Jr.; Sheikholislam, B.M.; Iwahashi-Hosoda, C.K.

    1982-01-01

    The capacities of normal and cystic fibrosis (CF) sera to bind to exogenous human (/sup 125/I)trypsin were compared. Sera from eight older CF patients bound significantly more exogenous human (/sup 125/I)trypsin than did sera from eight normal subjects (p less than 0.001). Disregarding the increased trypsin-binding (TB) of CF sera, serum immunoreactive trypsinogen (SIRT) levels were not detectable in these eight older CF patients. However, when SIRT levels were corrected for TB, four CF patients had normal SIRT concentrations and four had low but detectable SIRT levels. As compared to five normal newborns' sera, serum from a newborn with CF had normal TB and the SIRT levels were very high. In conclusion, increased TB in CF serum lowers results of SIRT assays. Therefore, unless SIRT levels are corrected for TB, results obtained from currently available SIRT kits may be invalid.

  12. Examination of the phenolic profile and antioxidant activity of the leaves of the Australian native plant Smilax glyciphylla.

    PubMed

    Huang, An-Cheng; Wilde, Amelie; Ebmeyer, Johanna; Skouroumounis, George K; Taylor, Dennis K

    2013-10-25

    Together with the sweet principle component glycyphyllin A (3), seven phenolic compounds including two new dihydrochalcone rhamnopyranosides, glycyphyllin B (1) and glycyphyllin C (2), and five known flavonoids, catechin (4), kaempferol-3-O-β-D-glucopyranoside (5), quercetin-3-O-β-D-glucopyranoside (6), kaempferol-3-O-β-neohesperidoside (7), and 2R,3R-dihydrokaempferol-3-O-β-D-glucopyranoside (8), have been isolated from the ethanolic extract of the leaves of Smilax glyciphylla for the first time. The structures of these compounds were characterized by spectroscopic methods including UV, MS, and 1D and 2D NMR. In vitro antioxidant capacity tests employing FRAP and DPPH assays indicated that 1, 4, and 6 exhibited potent antioxidant activity and are the key phenolics responsible for the antioxidant activity of the leaf extract of S. glyciphylla.

  13. In situ modification of activated carbons developed from a native invasive wood on removal of trace toxic metals from wastewater.

    PubMed

    de Celis, J; Amadeo, N E; Cukierman, A L

    2009-01-15

    Activated carbons were developed by phosphoric acid activation of sawdust from Prosopis ruscifolia wood, an indigenous invasive species of degraded lands, at moderate conditions (acid/precursor ratio=2, 450 degrees C, 0.5h). For in situ modification of their characteristics, either a self-generated atmosphere or flowing air was used. The activated carbons developed in the self-generated atmosphere showed higher BET surface area (2281m2/g) and total pore volume (1.7cm3/g) than those obtained under flowing air (1638m2/g and 1.3cm3/g). Conversely, the latter possessed a higher total amount of surface acidic/polar oxygen groups (2.2meq/g) than the former (1.5meq/g). To evaluate their metal sorption capability, adsorption isotherms of Cu(II) ion from model solutions were determined and properly described by the Langmuir model. Maximum sorption capacity (Xm) for the air-derived carbons (Xm=0.44mmol/g) almost duplicated the value for those obtained in the self-generated atmosphere (Xm=0.24mmol/g), pointing to a predominant effect of the surface functionalities on metal sequestering behaviour. The air-derived carbons also demonstrated a superior effectiveness in removing Cd(II) ions as determined from additional assays in equilibrium conditions. Accordingly, effective phosphoric acid-activated carbons from Prosopis wood for toxic metals removal from wastewater may be developed by in situ modification of their characteristics operating under flowing air.

  14. Time-course effects of centrally administered native urotensin-II on motor and cardioventilatory activity in trout.

    PubMed

    Lancien, F; Leprince, J; Mimassi, N; Mabin, D; Vaudry, H; Le Mével, J C

    2005-04-01

    Although in most vertebrate species urotensin-II (UII) is synthesized in neurons of the central nervous system, little is known regarding the physiological actions of UII in the brain. We have investigated the effects of intracerebroventricular (ICV) administration of synthetic trout UII (1, 5, and 50 pmol) on total motor activity (ACT), ventilatory frequency (VF), ventilatory amplitude (VA), and heart rate (HR) in the unanesthetized trout. ICV injection of UII increased ACT in a dose-dependent manner, and the maximal effect was observed at a dose of 5 pmol. At doses of 1 and 5 pmol, UII did not affect VF, VA, or HR. At the highest dose tested (50 pmol), UII not only increased ACT, but also significantly activated VF, VA, and HR. In contrast, ICV injection of synthetic trout angiotensin-II (5 pmol) did not produce any effect on ACT, VF, or VA, but sharply increased HR. These data provide the first evidence that UII can act centrally to induce motor activity in a nonmammalian vertebrate species.

  15. Influence of honey bee, Apis mellifera, hives and field size on foraging activity of native bee species in pumpkin fields.

    PubMed

    Artz, Derek R; Hsu, Cynthia L; Nault, Brian A

    2011-10-01

    The purpose of this study was to identify bee species active in pumpkin fields in New York and to estimate their potential as pollinators by examining their foraging activity. In addition, we examined whether foraging activity was affected by either the addition of hives of the honey bee, Apis mellifera L., or by field size. Thirty-five pumpkin (Cucurbita spp.) fields ranging from 0.6 to 26.3 ha, 12 supplemented with A. mellifera hives and 23 not supplemented, were sampled during peak flowering over three successive weeks in 2008 and 2009. Flowers from 300 plants per field were visually sampled for bees on each sampling date. A. mellifera, Bombus impatiens Cresson, and Peponapis pruinosa (Say) accounted for 99% of all bee visits to flowers. A. mellifera and B. impatiens visited significantly more pistillate flowers than would be expected by chance, whereas P. pruinosa showed no preference for visiting pistillate flowers. There were significantly more A. mellifera visits per flower in fields supplemented with A. mellifera hives than in fields not supplemented, but there were significantly fewer P. pruinosa visits in supplemented fields. The number of B. impatiens visits was not affected by supplementation, but was affected by number of flowers per field. A. mellifera and P. pruinosa visits were not affected by field size, but B. impatiens visited fewer flowers as field size increased in fields that were not supplemented with A. mellifera hives. Declining A. mellifera populations may increase the relative importance of B. impatiens in pollinating pumpkins in New York.

  16. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  17. Content in Native Literature Programs.

    ERIC Educational Resources Information Center

    Grant, Agnes

    Including Native literature in school curricula is an important way of enhancing the Native student's self-concept and providing accurate Native cultural knowledge to Native and non-Native students alike. Nevertheless, Canadian school literature programs generally contain neither contemporary nor traditional Native literature. Some programs…

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

    SciTech Connect

    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.

  19. Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein.

    PubMed

    Luo, M; Weinstein, J D; Walker, C J

    1999-12-01

    Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.

  20. Kinetic properties of three isoforms of trypsin isolated from the pyloric caeca of chum salmon (Oncorhynchus keta).

    PubMed

    Toyota, Eiko; Iyaguchi, Daisuke; Sekizaki, Haruo; Itoh, Kunihiko; Tanizawa, Kazutaka

    2007-09-01

    Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K(m) values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 degrees C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k(cat), of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k(cat)/K(m)) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K(m) and k(cat) values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K(m) values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k(cat) values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k(cat)/K(m)) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k(cat)/K(m) values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k(cat)/K(m) values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

  1. Substituted isatoic anhydrides: selective inactivators of trypsin-like serine proteases.

    PubMed

    Gelb, M H; Abeles, R H

    1986-04-01

    Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituents irreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride. 7-(Aminomethyl)-1-benzylisatoic anhydride was prepared and was a selective inactivator of thrombin; thus it is possible to prepare derivatives of isatoic anhydride that are highly enzyme selective without attaching peptide recognition structures.

  2. Soccer activity profile of altitude versus sea-level natives during acclimatisation to 3600 m (ISA3600)

    PubMed Central

    Aughey, Robert J; Hammond, Kristal; Varley, Matthew C; Schmidt, Walter F; Bourdon, Pitre C; Buchheit, Martin; Simpson, Ben; Garvican-Lewis, Laura A; Kley, Marlen; Soria, Rudy; Sargent, Charli; Roach, Gregory D; Claros, Jesus C Jimenez; Wachsmuth, Nadine; Gore, Christopher J

    2013-01-01

    Objectives We investigated the effect of high altitude on the match activity profile of elite youth high altitude and sea level residents. Methods Twenty Sea Level (Australian) and 19 Altitude-resident (Bolivian) soccer players played five games, two near sea level (430 m) and three in La Paz (3600 m). Match activity profile was quantified via global positioning system with the peak 5 min period for distance ((D5peak)) and high velocity running (>4.17 m/s, HIVR5peak); as well as the 5 min period immediately subsequent to the peak for both distance (D5sub) and high-velocity running (HIVR5sub) identified using a rolling 5 min epoch. The games at 3600 m were compared with the average of the two near sea-level games. Results The total distance per minute was reduced by a small magnitude in the first match at altitude in both teams, without any change in low-velocity running. There were variable changes in HiVR, D5peak and HiVR5peak from match to match for each team. There were within-team reductions in D5peak in each game at altitude compared with those at near sea level, and this reduction was greater by a small magnitude in Australians than Bolivians in game 4. The effect of altitude on HiVR5peak was moderately lower in Australians compared with Bolivians in game 3. There was no clear difference in the effect of altitude on maximal accelerations between teams. Conclusions High altitude reduces the distance covered by elite youth soccer players during matches. Neither 13 days of acclimatisation nor lifelong residence at high altitude protects against detrimental effects of altitude on match activity profile. PMID:24282196

  3. Bestrophin-Encoded Ca2+-Activated Cl− Channels Underlie a Current with Properties Similar to the Native Current in the Moth Spodoptera littoralis Olfactory Receptor Neurons

    PubMed Central

    Demondion, Elodie; Bozzolan, Françoise; Debernard, Stéphane; Lucas, Philippe

    2012-01-01

    Responses of insect olfactory receptor neurons (ORNs) involve an entry of Ca2+ through olfactory heterodimeric receptor complexes. In moths, the termination of ORN responses was found to strongly depend on the external Ca2+ concentration through the activation of unknown Ca2+-dependent Cl− channels. We thus investigated the molecular identity of these Cl− channels. There is compelling evidence that bestrophins form Cl− channels when expressed in heterologous systems. Here we provide evidence that antennae of the moth Spodoptera littoralis express three transcripts encoding proteins with hallmarks of bestrophins. One of these transcripts, SlitBest1b, is expressed in ORNs. The heterologous expression of SlitBest1b protein in CHO-K1 cells yielded a Ca2+-activated Cl− current that shares electrophysiological properties with the native Ca2+-activated Cl− current of ORNs. Both currents are anionic, present similar dependence on the intracellular Ca2+ concentration, partly inactivate over time, have the same anion permeability sequence, the same sequence of inhibitory efficiency of blockers, the same almost linear I–V relationships and finally both currents do not depend on the cell volume. Therefore, our data suggest that SlitBest1b is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel and is likely to constitute part of the insect olfactory transduction pathway. A different function (e.g. regulation of other proteins, maintenance of the anionic homeostasis in the sensillar lymph) and a different role (e.g. involvement in the olfactory system development) cannot be excluded however. PMID:23300744

  4. Auditory-motor interactions for the production of native and non-native speech.

    PubMed

    Parker Jones, Oiwi; Seghier, Mohamed L; Kawabata Duncan, Keith J; Leff, Alex P; Green, David W; Price, Cathy J

    2013-02-06

    During speech production, auditory processing of self-generated speech is used to adjust subsequent articulations. The current study investigated how the proposed auditory-motor interactions are manifest at the neural level in native and non-native speakers of English who were overtly naming pictures of objects and reading their written names. Data were acquired with functional magnetic resonance imaging and analyzed with dynamic causal modeling. We found that (1) higher activity in articulatory regions caused activity in auditory regions to decrease (i.e., auditory suppression), and (2) higher activity in auditory regions caused activity in articulatory regions to increase (i.e., auditory feedback). In addition, we were able to demonstrate that (3) speaking in a non-native language involves more auditory feedback and less auditory suppression than speaking in a native language. The difference between native and non-native speakers was further supported by finding that, within non-native speakers, there was less auditory feedback for those with better verbal fluency. Consequently, the networks of more fluent non-native speakers looked more like those of native speakers. Together, these findings provide a foundation on which to explore auditory-motor interactions during speech production in other human populations, particularly those with speech difficulties.

  5. Real-time electro-mechano-acoustic imaging for monitoring interactions between trypsin and different inhibitors in articular cartilage.

    PubMed

    Zheng, Yong-Ping; Wang, Qing; Butt, Yoki Kwok Chu

    2011-03-01

    The purpose of this study was to observe the real-time interactions between trypsin and various inhibitors in articular cartilage in vitro using a novel electro-mechano-acoustic imaging method. Monitored in real-time, articular cartilage specimens from bovine patellae were first treated with trypsin to reach half proteoglycan depletion (Phase I), then the trypsin solution was replaced with (i) physiological saline buffer (PS), (ii) fetal bovine serum (FBS), (iii) protease inhibitor cocktail (PI) and (iv) 10% formalin (F), respectively, to observe their effects on residual digestion (Phase II). Ultrasound radio frequency signals from the articular cartilage were used to form a M-mode image, where the interface between trypsin digested and intact cartilage tissues could be observed with an additional echo generated. The inhibition time, the digestion depth and digestion fraction were measured for each specimen. The results showed that the dilution of trypsin using saline solution was not sufficient to stop the enzyme action instantly. Although groups FBS and PI had a similar inhibition time of approximately 1.5 h, their digestion depth was obviously different (0.25±0.03 and 0.06±0.06 mm, respectively). In contrast, formalin only took <30 min to stop the trypsin digestion with almost no further digestion. The results demonstrated that the current system was capable of monitoring the trypsin digestion and inhibition process in real time. Also, different chemicals affected the residual trypsin digestion to different degrees.

  6. Calcium addition at the Hubbard Brook Experimental Forest increases sugar storage, antioxidant activity and cold tolerance in native red spruce (Picea rubens).

    PubMed

    Halman, Joshua M; Schaberg, Paul G; Hawley, Gary J; Eagar, Christopher

    2008-06-01

    In fall (November 2005) and winter (February 2006), we collected current-year foliage of native red spruce (Picea rubens Sarg.) growing in a reference watershed and in a watershed treated in 1999 with wollastonite (CaSiO(3), a slow-release calcium source) to simulate preindustrial soil calcium concentrations (Ca-addition watershed) at the Hubbard Brook Experimental Forest (Thornton, NH). We analyzed nutrition, soluble sugar concentrations, ascorbate peroxidase (APX) activity and cold tolerance, to evaluate the basis of recent (2003) differences between watersheds in red spruce foliar winter injury. Foliar Ca and total sugar concentrations were significantly higher in trees in the Ca-addition watershed than in trees in the reference watershed during both fall (P=0.037 and 0.035, respectively) and winter (P=0.055 and 0.036, respectively). The Ca-addition treatment significantly increased foliar fructose and glucose concentrations in November (P=0.013 and 0.007, respectively) and foliar sucrose concentrations in winter (P=0.040). Foliar APX activity was similar in trees in both watersheds during fall (P=0.28), but higher in trees in the Ca-addition watershed during winter (P=0.063). Cold tolerance of foliage was significantly greater in trees in the Ca-addition watershed than in trees in the reference watershed (P<0.001). Our results suggest that low foliar sugar concentrations and APX activity, and reduced cold tolerance in trees in the reference watershed contributed to their high vulnerability to winter injury in 2003. Because the reference watershed reflects forest conditions in the region, the consequences of impaired physiological function caused by soil Ca depletion may have widespread implications for forest health.

  7. Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis.

    PubMed

    Fan, Huizhi; Bao, Huimin; Zhang, Luyan; Chen, Gang

    2011-08-01

    Trypsin was covalently immobilized on poly(urea-formaldehyde)-coated fiberglass cores based on the condensation reaction between poly(urea-formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin-immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core-changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI-TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12-h in-solution digestion.

  8. Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

    PubMed

    Xu, Xiao-yan; Nie, Xiao-cui; Ma, Hai-ying; Song, Guo-qing; Zhang, Xiao-tong; Jin, Yu-nan; Yu, Yan-qiu

    2015-04-01

    Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group.

  9. Impact of mechanical stress on ion transport in native lung epithelium (Xenopus laevis): short-term activation of Na+, Cl (-) and K+ channels.

    PubMed

    Bogdan, Roman; Veith, Christine; Clauss, Wolfgang; Fronius, Martin

    2008-09-01

    Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I (SC): 24 +/- 1%, n = 152), slightly decreased the transepithelial resistance (R (T): 7 +/- 2%, n = 152), but increased the apical membrane capacitance (C (M): 16 +/- 6%, n = 9). The pressure-induced effect was sensitive to Na+ (amiloride), Cl(-) (DIDS, NFA, NPPB) and K+ channel blockers (Ba2+), glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na+, Cl(-), and K+ conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.

  10. From acute to chronic pancreatitis: the role of mutations in the pancreatic secretory trypsin inhibitor gene.

    PubMed

    Hirota, Masahiko; Kuwata, Kinuko; Ohmuraya, Masaki; Ogawa, Michio

    2003-03-01

    Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed the hypothesis that, if the function of the PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis and we performed a mutational analysis of the PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were thought to be associated with a predisposition to pancreatitis. The N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of the recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations, rather than N34S itself (IVS1-37T>C + N34S + IVS3-69insTTTT complex), may be associated with the decreased function of the PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intra-molecular or inter-molecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand-new findings in PSTI knockout mice, suggest that the genetic mutation of the PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

  11. Characterization and expression analysis of a trypsin-like serine protease from planarian Dugesia japonica.

    PubMed

    Zhou, Luming; Wu, Suge; Liu, Dianchen; Xu, Bo; Zhang, Xiufang; Zhao, Bosheng

    2012-06-01

    Trypsin-like serine proteases are involved in large number of processes, especially in digestive degradation and immune responses. Here, we identify the characterization of a trypsin-like serine protease in planarian, Djtry, which interestingly has the incompletely conserved catalytic triad (K, D, and S). Phylogenetic analysis suggests that Djtry is an ancient type of trypsin-like serine proteases. The spatial and temporal expression patterns of Djtry are shown during regenerating and embryonic development by whole-mount in situ hybridization. Djtry is found to display a tissue specific expression pattern, with a predominant expression detected in whole gut region of intact and regenerating planarian. While the tissue- and stage-specific expression patterns during the embryonic development imply the roles of Djtry involve in yolk degradation and gut formation. Quantitative real-time PCR was carried out to analyze the function of this protease in vivo after planarians were stimulated to a bacterial challenge and food. The results showed that Djtry increased after a bacterial challenge and was basically stable for food. Therefore, the trypsin-like serine protease might be involved in the innate defense reactions against bacterial infection.

  12. Damage and repair of the peripheral myelin sheath and node of Ranvier after treatment with trypsin.

    PubMed

    Yu, R C; Bunge, R P

    1975-01-01

    Cultures of whole fetal rat sensory ganglia which had matured and myelinated in culture were treated for 1-3 h with a pulse of 0.2% trypsin. The tissue was observed during the period of treatment and during subsequent weeks using both light and electron microscopy. Within minutes after trypsin addition the matrix of the culture was altered and the nerve fascicles loosened. Progressive changes included the retraction of Schwann cell processes from the nodal region the detachment of the myelin-related paranodal Schwann cell loops from the axon, and lengthening of the nodal region as the axon was bared. The retraction of myelin from nodal stabilized several hours after trypsin withdrawal. Breakdown of the altered myelin segments was rare. There were no discernable changes in neurons or their processes after this exposure to trypsin. The partial repair which occured over a period of several weeks included the reattachment of paranodal Schwann cell loops to the axolemma and the insertion of new myelin segments where a substantial length of axolemma had been bared. The significance of these observations to the characterization of the Schwann cell-axolemmal junctions on myelinated nerve fibers is discussed. The dramatic degree of myelin change that can occur without concomitant myelin breakdown is particularly noted, as is the observation that these altered myelin segments are, in part, repaired.

  13. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... system. 866.5890 Section 866.5890 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An...

  14. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... system. 866.5890 Section 866.5890 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An...

  15. Trypsinization of monkey-kidney tissue: an automatic method for the preparation of cell suspensions*

    PubMed Central

    Rappaport, Catherine

    1956-01-01

    A study of some of the factors which influenced the rate and amount of cells released from tissue fragments during trypsinization led to a revision of the method described by Youngner for monkey kidney. The revision includes the use of a glass mixing-chamber and magnetic stirrer in place of the Waring blendor. Simpler to use, the revised method has been found to yield, consistently, about 7 × 10 7 cells per g of kidney tissue, or from two to three times more than that obtained by the earlier method. The revised method may be done either manually or automatically. A simple glass apparatus which automatically regulates the continuous addition of trypsin and removal of cell suspension during trypsinization has been developed. It operates reliably over a threefold volume range and a varying flow rate. The yield of cells per gram of tissue treated in the automatic trypsinizer is about 30% greater than when the change of fluids is done manually. ImagesFIG. 1FIG. 3 PMID:13329843

  16. Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis.

    PubMed

    Lin, Shuang; Yun, Dong; Qi, Dawei; Deng, Chunhui; Li, Yan; Zhang, Xiangmin

    2008-03-01

    In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI-MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins ( p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.

  17. Traditional Native Poetry.

    ERIC Educational Resources Information Center

    Grant, Agnes

    1985-01-01

    While Native myths and legends were educational tools to transmit tribal beliefs and history, traditional American Indian poetry served a ritualistic function in everyday life. Few traditional Native songs, which all poems were, survive; only Mayan and Aztec poems were written, and most of these were burned by a Spanish bishop. In addition, many…

  18. Native American Preparatory School.

    ERIC Educational Resources Information Center

    Native American Preparatory School, Rowe, NM.

    This booklet provides information on the Native American Preparatory School, a residential secondary school in Rowe, New Mexico, for high-achieving Native American students. The school sponsors two programs: a 5-week rigorously academic summer school for junior high school students and, beginning in fall 1995, a 4-year college preparatory program.…

  19. Native American Entrepreneurship. Digest.

    ERIC Educational Resources Information Center

    Seymour, Nicole

    Although Native Americans have owned and started the fewest small businesses of all U.S. minority groups, entrepreneurship is considered to be an efficient tool for alleviating their economic problems. Barriers to Native American entrepreneurship include poverty, scarce start-up capital, poor access to business education and technical assistance,…

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

    PubMed

    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.

  1. Native SAD is maturing

    PubMed Central

    Rose, John P.; Wang, Bi-Cheng; Weiss, Manfred S.

    2015-01-01

    Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow ‘highly accurate data’ to be routinely collected. Today, native SAD sits on the verge of becoming a ‘first-choice’ method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development. PMID:26175902

  2. Recognition and stabilization of a unique CPRI--structural motif in cucurbitaceae family trypsin inhibitor peptides: molecular dynamics based homology modeling using the X-ray structure of MCTI-II.

    PubMed

    Chakraborty, S; Haldar, U; Bera, A K; Pal, A K; Bhattacharya, S; Ghosh, S; Mukhopadhyay, B P; Banerjee, A

    2001-02-01

    The high resolution crystallographic structure of MCTI-II complexed with beta trypsin (PDB entry 1MCT) was used to model the corresponding structures of the six inhibitor peptides belonging to Cucurbitaceae family (MCTI-I, LA-1, LA-2, CMTI-I, CMTI-III, CMTI-IV). Two model inhibitors, LA-1 and LA-2 were refined by molecular dynamics to estimate the average solution structure. The difference accessible surface area (DASA) study of the inhibitors with and without trypsin revealed the Arginine and other residues of the inhibitors which bind to trypsin. The hydration dynamics study of LA1 and LA2 also confirm the suitability of water molecules at the active Arg site. Moreover, the presence of a unique 3D-structural motif comprises with the four CPRI residues from the amino terminal is thought to be conserved in all the six studied inhibitors, which seems essential for the directional fixation for proper complexation of the Arg (5) residue towards the trypsin S1-binding pocket. The role of the disulphide linkage in the geometrical stabilization of CPRI (Cysteine, Proline, Arginine, Isoleucine) motif has also been envisaged from the comparative higher intra molecular Cys (3) -Cys (20) disulphide dihedral energies.

  3. Chinese tallow trees (Triadica sebifera) from the invasive range outperform those from the native range with an active soil community or phosphorus fertilization.

    PubMed

    Zhang, Ling; Zhang, Yaojun; Wang, Hong; Zou, Jianwen; Siemann, Evan

    2013-01-01

    Two mechanisms that have been proposed to explain success of invasive plants are unusual biotic interactions, such as enemy release or enhanced mutualisms, and increased resource availability. However, while these mechanisms are usually considered separately, both may be involved in successful invasions. Biotic interactions may be positive or negative and may interact with nutritional resources in determining invasion success. In addition, the effects of different nutrients on invasions may vary. Finally, genetic variation in traits between populations located in introduced versus native ranges may be important for biotic interactions and/or resource use. Here, we investigated the roles of soil biota, resource availability, and plant genetic variation using seedlings of Triadica sebifera in an experiment in the native range (China). We manipulated nitrogen (control or 4 g/m(2)), phosphorus (control or 0.5 g/m(2)), soil biota (untreated or sterilized field soil), and plant origin (4 populations from the invasive range, 4 populations from the native range) in a full factorial experiment. Phosphorus addition increased root, stem, and leaf masses. Leaf mass and height growth depended on population origin and soil sterilization. Invasive populations had higher leaf mass and growth rates than native populations did in fresh soil but they had lower, comparable leaf mass and growth rates in sterilized soil. Invasive populations had higher growth rates with phosphorus addition but native ones did not. Soil sterilization decreased specific leaf area in both native and exotic populations. Negative effects of soil sterilization suggest that soil pathogens may not be as important as soil mutualists for T. sebifera performance. Moreover, interactive effects of sterilization and origin suggest that invasive T. sebifera may have evolved more beneficial relationships with the soil biota. Overall, seedlings from the invasive range outperformed those from the native range, however

  4. Signalling pathways activated by 5-HT(1B)/5-HT(1D) receptors in native smooth muscle and primary cultures of rabbit renal artery smooth muscle cells.

    PubMed

    Hinton, J M; Hill, P; Jeremy, J; Garland, C

    2000-01-01

    The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT(1B)/5-HT(1D) receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 microM), and prestimulated with 20 mM K(+) Krebs buffer, 5-HT and CP 93,129, a 5-HT(1B) receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT(1B)/5-HT(1D) receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT(1B) receptor and the 5-HT(1D) receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 +/- 3.33 and 52.65 +/- 5.34% from basal with 10 microM 5-HT, respectively). The effect of 10 microM 5-HT on cAMP was increased in the presence of 20 mM K(+) (reduced by 82.50 +/- 2.50 and 87.54 +/- 3.97%, respectively). In intact arteries, contraction through 5-HT(1B)/5-HT(1D) receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U0126). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K(+) was present at the onset of stimulation. These data provide the first direct evidence that 5-HT(1B)/5-HT(1B) receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a

  5. Spatial prediction of habitat overlap of introduced and native thistles to identify potential areas of nontarget activity of biological control agents.

    PubMed

    Wiggins, G J; Grant, J F; Lambdin, P L; Ranney, Jack W; Wilkerson, J B; van Manen, F T

    2010-12-01

    Nontarget feeding of Rhinocyllus conicus Fröelich and Trichosirocalus horridus (Panzer) on native North American thistles in the genus Cirsium has been documented. Some species of these native thistles have shown greater infestation levels of R. conicus in populations that are in close proximity to the target plant species, Carduus nutans L. In 2005 a study was initiated to identify areas of potential nontarget feeding by R. conicus and T. horridus on thistle species by predicting habitats of two known introduced hosts [C. nutans and Cirsium vulgare (Savi) Tenore] and two native species [Cirsium carolinianum (Walter) Fernald and Schubert and C. discolor (Muhlenberg ex Willdenow) Sprengel] using Mahalanobis distance (D(2)). Cumulative frequency graphs showed that the D(2) models for all four plant species effectively identified site conditions that contribute to the presence of the respective species. Poisson regression showed an association between D(2) values and plant counts at field-test sites for C. nutans and C. carolinianum. However, negative binomial regression detected no association between D(2) values and plant counts for C. discolor or C. vulgare. Chi-square analysis indicated associations between both weevil species and sites where C. vulgare and Carduus nutans were found, but not between the weevil and native thistle species. Habitats of C. nutans and Cirsium carolinianum overlapped in ≈12% of the study area. Data-based habitat models may provide a powerful tool for land managers and scientists to monitor native plant populations for nontarget feeding by introduced biological control agents.

  6. The on-bead digestion of protein corona on nanoparticles by trypsin immobilized on the magnetic nanoparticle.

    PubMed

    Hu, Zhengyan; Zhao, Liang; Zhang, Hongyan; Zhang, Yi; Wu, Ren'an; Zou, Hanfa

    2014-03-21

    Proteins interacting with nanoparticles would form the protein coronas on the surface of nanoparticles in biological systems, which would critically impact the biological identities of nanoparticles and/or result in the physiological and pathological consequences. The enzymatic digestion of protein corona was the primary step to achieve the identification of protein components of the protein corona for the bottom-up proteomic approaches. In this study, the investigation on the tryptic digestion of protein corona by the immobilized trypsin on a magnetic nanoparticle was carried out for the first time. As a comparison with the usual overnight long-time digestion and the severe self-digestion of free trypsin, the on-bead digestion of protein corona by the immobilized trypsin could be accomplished within 1h, along with the significantly reduced self-digestion of trypsin and the improved reproducibility on the identification of proteins by the mass spectrometry-based proteomic approach. It showed that the number of identified bovine serum (BS) proteins on the commercial Fe3O4 nanoparticles was increased by 13% for the immobilized trypsin with 1h digestion as compared to that of using free trypsin with even overnight digestion. In addition, the on-bead digestion of using the immobilized trypsin was further applied on the identification of human plasma protein corona on the commercial Fe3O4 nanoparticles, which leads the efficient digestion of the human plasma proteins and the identification of 149 human plasma proteins corresponding to putative critical pathways and biological processes.

  7. History of NASA/Native People Native Homelands Initiative

    NASA Technical Reports Server (NTRS)

    Maynard, Nancy

    2000-01-01

    This workshop is one of the follow-on local assessment activities from the US National Assessment on the Impact of Climate Change on the US. N. Maynard (for NASA) helped create and get under way an initiative which brought together climate change scientists from around the US with Native Americans to bring together classic Western European scientists with knowledge from native peoples - from such sources as oral histories of drought, major fires, etc. The purpose of this was to encourage not only joint science but also bring NASA resources and education materials to Tribal schools and encourage joint preparation of educational and training materials. N. Maynard's talk will provide history of that process and discuss possible ways to collaborate in the future, building on this effort.

  8. Improvement of the CuZn-superoxide dismutase enzyme activity and stability as a therapeutic agent by modification with polysialic acids.

    PubMed

    Wu, Jian Rong; Lin, Yi; Zheng, Zhi Yong; Lin, Chi Chung; Zhan, Xiao Bei; Shen, Ying Qiang

    2010-12-01

    The optimal process for the polysialylation reaction was as follows: polysialicacid (PSA) was activated by periodate oxidation, then coupled to CuZn superoxide dismutase (SOD) with a PSA:SOD molar ratio of 40:1 for 24 h. The resulting polysialylated protein contained 3.9 ± 0.3 mol PSA per mol SOD. SDS-PAGE and atomic force microscopy revealed that the molecular weight of polysialylated SOD was about 90-100 kDa. The average size was 10-15 nm, about four-fold of the native enzyme. Compared to the native enzyme, the activity and stability of the polysialylated SOD, as well as resistance to heat, acid, alkali and proteases present in human digestive system such as pepsin and trypsin, were improved significantly as therapeutic agent.

  9. The Native Comic Book Project: Native Youth Making Comics and Healthy Decisions

    PubMed Central

    Montgomery, Michelle; Manuelito, Brenda; Nass, Carrie; Chock, Tami; Buchwald, Dedra

    2015-01-01

    Background American Indians and Alaska Natives have traditionally used stories and drawings to positively influence the well-being of their communities. Objectives The objective of this study was to describe the development of a curriculum that trains Native youth leaders to plan, write, and design original comic books to enhance healthy decision making. Methods Project staff developed the Native Comic Book Project by adapting Dr. Michael Bitz’s Comic Book Project to incorporate Native comic book art, Native