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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. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  3. Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti.

    PubMed

    Barillas-Mury, C V; Noriega, F G; Wells, M A

    1995-02-01

    Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility. PMID:7711754

  4. Specific activation of the thyrotropin receptor by trypsin.

    PubMed

    Van Sande, J; Massart, C; Costagliola, S; Allgeier, A; Cetani, F; Vassart, G; Dumont, J E

    1996-05-31

    The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain. PMID:8807635

  5. A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

    PubMed

    Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

    2014-08-01

    Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM. PMID:23859150

  6. 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.

  7. Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin

    PubMed Central

    2013-01-01

    Background Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2). Results During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2. Conclusion These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2. PMID:23870109

  8. Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

    PubMed Central

    Cook, R R; Powers, J C

    1983-01-01

    Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes. PMID:6360155

  9. Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti.

    PubMed

    Pontual, Emmanuel Viana; Napoleão, Thiago Henrique; Dias de Assis, Caio Rodrigo; de Souza Bezerra, Ranilson; Xavier, Haroudo Satiro; Navarro, Daniela Maria do Amaral Ferraz; Coelho, Luana Cassandra Breitenbach Barroso; Paiva, Patrícia Maria Guedes

    2012-03-01

    Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality. PMID:22392801

  10. Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.

    PubMed Central

    Belham, C M; Tate, R J; Scott, P H; Pemberton, A D; Miller, H R; Wadsworth, R M; Gould, G W; Plevin, R

    1996-01-01

    We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species. PMID:9003384

  11. 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.

  12. 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.

  13. 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

  14. A Trypsin Inhibitor from Clitoria fairchildiana Cotyledons is Active Against Digestive Enzymes of Aedes aegypti Larvae.

    PubMed

    de Oliveira, Lucilene O; Fernandes, Kátia V S; Pádua, Dayanni de Souza; Carvalho, André de O; Lemos, Francisco J A; Gomes, Valdirene M; Oliveira, Antônia E A; Ferreira, André T da Silva; Perales, Jonas

    2015-01-01

    Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method. PMID:26156641

  15. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

    PubMed Central

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J.; Wubbolts, Richard W.; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.

    2014-01-01

    ABSTRACT Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  16. Nutritional significance of a rice bran concentrate with trypsin inhibitor activity.

    PubMed

    Maki, Z; Tashiro, M

    1983-06-01

    A rice bran protein concentrate (RBPC) was prepared from de-fatted rice bran by extraction with a 1% sodium chloride solution and by acetone-precipitation. This protein concentrate contained 45% protein, which was as good as casein in terms of protein quality being judged from the results of amino acid analysis. On the other hand, RBPC possessed the trypsin inhibitor activity corresponding to the complete inhibition of about 6 mg of bovine trypsin per 1 g of dry material. The activity was, however, completely destroyed by autoclaving RBPC for 30 min at 121 degrees C. In vitro digestion tests showed that RBPC was easily digested by pepsin but was resistant to the attack by trypsin, compared with autoclaved RBPC. Concerning in vivo digestion, however, there was no significant difference in apparent nitrogen digestibility between RBPC and the heated RBPC. In growth experiments with weanling rats fed a 10% level of protein diet, growth depression and the tendency of slight pancreatic hypertrophy were observed in rats receiving a RBPC diet. It is presumed that one of the reasons which explains these phenomena is the presence of trypsin inhibitor in RBPC. PMID:6619992

  17. Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis.

    PubMed

    Lemeire, Els; Borovsky, Dov; Van Camp, John; Smagghe, Guy

    2008-12-01

    Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, down-regulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and development. PMID:18949805

  18. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

    PubMed

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-07-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  19. 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

  20. Redesigning Trypsin: Alteration of Substrate Specificity

    NASA Astrophysics Data System (ADS)

    Craik, Charles S.; Largman, Corey; Fletcher, Thomas; Roczniak, Steven; Barr, Philip J.; Fletterick, Robert; Rutter, William J.

    1985-04-01

    A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

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

    PubMed Central

    Samac, Deborah; Storey, Richard

    1981-01-01

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

  2. 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. PMID:27507460

  3. Catalytic activity of trypsin entrapped in electrospun poly(ϵ-caprolactone) nanofibers.

    PubMed

    Pinto, Susana C; Rodrigues, Ana R; Saraiva, Jorge A; Lopes-da-Silva, José A

    2015-11-01

    Trypsin was successfully entrapped in situ into nanofibers of poly(ϵ-caprolactone) (PCL) prepared by electrospinning. The spinning dope was an emulsion consisting of an aqueous phase with the solubilized enzyme in a pH buffer plus an oil phase of the polymer solubilized in chloroform (CF)/dimethylformamide (DMF). The optimized materials were composed by random arrays of bead-free fibers with outer diameters in the range 110-180 nm without showing core-shell structure. The fiber size and morphology, membrane porosity and surface properties were shown to be influenced by the polymer concentration and the composition ratio of the solvent mixture, and also by the presence of the enzyme. The activity of the immobilized trypsin was studied toward both a low-molecular weight synthetic substrate (BAPNA) and a protein (casein). Fluorescence microscopy, the increasing hydrophilicity of the fibrous membrane and the observed catalytic activity confirmed the entrapment of the enzyme into the PCL nanofibers. The best activity retention (∼66% toward BAPNA) was achieved using 0.20 g/mL PCL in CF/DMF [75:25], with trypsin in an aqueous buffer at pH 7.1 in the presence of benzamidine and Span80. The immobilized enzyme showed satisfactory operational stability retaining ∼59% of its initial activity after five reaction cycles. Compared with the free enzyme, the storage (at 4 °C) and thermal stability of the immobilized enzyme were highly improved. The retained catalytic activity and the observed reusability can be explained by a heterogeneous distribution of the enzyme within the polymer fiber influenced by the electrostatic field during the electrospinning process, enabling a preferential location near the fiber surface but simultaneously assuring minimal leaching out during operations. Results suggest that trypsin-PCL fibrous membranes may be useful for concomitant proteolytic and separation commercial applications. PMID:26320709

  4. Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization.

    PubMed

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

    2006-05-01

    Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes. PMID:16500127

  5. The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases

    PubMed Central

    2014-01-01

    Background Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species. Methods In the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity. Results The combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines. Conclusion These results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus

  6. 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. PMID:24047891

  7. 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

  8. [Effect of the interphase surface charge on the structure and activity of trypsin in inverted micelles].

    PubMed

    Valiullina, Iu A; Stupishina, E A; Vylegzhanina, N N; Idiiatullin, B Z; Zuev, Iu F

    2008-01-01

    The effect of the sign and value of the charge of the interphase surface on the catalytic activity of trypsin in systems of inverted mycelles was investigated. n-Butanol was used for the modification of the phase interface in dispersions of inverted mycelles based on anionic sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cationic cetyltrimethylammonium bromide (CTAB). A direct correlation between changes in the state of inverted mycelles and the structure of solubilized enzyme under the action of butanol was obtained. It was shown that the enzyme activity is determined by the quantity of butanol solubilized by the inverted mycelles. PMID:18672691

  9. Quantitative structure-activity relationships for organophosphates binding to trypsin and chymotrypsin.

    PubMed

    Ruark, Christopher D; Hack, C Eric; Robinson, Peter J; Gearhart, Jeffery M

    2011-01-01

    Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX) do not react solely with acetylcholinesterase (AChE). Evidence suggests that cholinergic-independent pathways over a wide range are also targeted, including serine proteases. These proteases comprise nearly one-third of all known proteases and play major roles in synaptic plasticity, learning, memory, neuroprotection, wound healing, cell signaling, inflammation, blood coagulation, and protein processing. Inhibition of these proteases by OP was found to exert a wide range of noncholinergic effects depending on the type of OP, the dose, and the duration of exposure. Consequently, in order to understand these differences, in silico biologically based dose-response and quantitative structure-activity relationship (QSAR) methodologies need to be integrated. Here, QSAR were used to predict OP bimolecular rate constants for trypsin and α-chymotrypsin. A heuristic regression of over 500 topological/constitutional, geometric, thermodynamic, electrostatic, and quantum mechanical descriptors, using the software Ampac 8.0 and Codessa 2.51 (SemiChem, Inc., Shawnee, KS), was developed to obtain statistically verified equations for the models. General models, using all data subsets, resulted in R(2) values of .94 and .92 and leave-one-out Q(2) values of 0.9 and 0.87 for trypsin and α-chymotrypsin. To validate the general model, training sets were split into independent subsets for test set evaluation. A y-randomization procedure, used to estimate chance correlation, was performed 10,000 times, resulting in mean R(2) values of .24 and .3 for trypsin and α-chymotrypsin. The results show that these models are highly predictive and capable of delineating the complex mechanism of action between OP and serine proteases, and ultimately, by applying this approach to other OP enzyme reactions such as AChE, facilitate the development of biologically based

  10. Vegetative Storage Protein in Litchi chinensis, a Subtropical Evergreen Fruit Tree, Possesses Trypsin Inhibitor Activity

    PubMed Central

    Tian, Wei-Min; Peng, Shi-Qing; Wang, Xu-Chu; Shi, Min-Jing; Chen, Yue-Yi; Hu, Zheng-Hai

    2007-01-01

    Background and Aims Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. Key Results The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. Conclusions Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated. PMID:17913726

  11. Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.

    PubMed

    Wen, Yingxia; Palladino, Giuseppe; Xie, Yuhong; Ferrari, Annette; Ma, Xiuwen; Han, Liqun; Dormitzer, Philip R; Settembre, Ethan C

    2016-06-17

    Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA. PMID:27154389

  12. 13C-egg white breath test: a non-invasive test of pancreatic trypsin activity in the small intestine

    PubMed Central

    Evenepoel, P; Hiele, M; Geypens, B; Geboes, K; Rutgeerts, P; Ghoos, Y

    2000-01-01

    BACKGROUND—The recent availability of egg white protein highly enriched with 13C has allowed breath test technology to be adapted for the study of protein digestion and absorption. Pancreatic trypsin is considered to be the key enzyme in the proteolytic cascade.
AIM—To evaluate trypsin activity in the small intestine of healthy volunteers and patients with pancreatic disease by a recently developed 13C-egg white breath test.
METHODS—A total of 48 healthy volunteers and 30 patients with pancreatic disease were studied after ingestion of a test meal consisting of 22 g 13C-labelled egg protein. Breath samples were taken before and after ingestion of the meal and analysed for 13CO2 concentration. Moreover, pancreatic trypsin output after maximal stimulation was measured in 13 patients and nine healthy volunteers.
RESULTS—The six hour cumulative 13CO2 excretion in breath was significantly lower in patients than controls (mean (SEM): 6.23 (0.82)% v 19.16 (0.58)%, p<0.0001). An excellent correlation was found between the six hour cumulative 13CO2 excretion and trypsin activity after maximal pancreatic stimulation.
CONCLUSION—The non-invasive 13C-egg white breath test is promising as an indirect pancreatic proteolytic function test.


Keywords: breath test; pancreatic disease; trypsin; protein; assimilation PMID:10601055

  13. Cleaning out the Litterbox of Proteomic Scientists’ Favorite Pet: Optimized Data Analysis Avoiding Trypsin Artifacts

    PubMed Central

    2016-01-01

    Chemically modified trypsin is a standard reagent in proteomics experiments but is usually not considered in database searches. Modification of trypsin is supposed to protect the protease against autolysis and the resulting loss of activity. Here, we show that modified trypsin is still subject to self-digestion, and, as a result, modified trypsin-derived peptides are present in standard digests. We depict that these peptides commonly lead to false-positive assignments even if native trypsin is considered in the database. Moreover, we present an easily implementable method to include modified trypsin in the database search with a minimal increase in search time and search space while efficiently avoiding these false-positive hits. PMID:26938934

  14. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  15. Trypsin-like proteinase and its endogenous inhibitor from Yersinia pseudotuberculosis. Biological activity.

    PubMed

    Burtseva, T I; Loenko, Y N

    1999-09-01

    A trypsin-like proteinase (YPTP) and its endogenous inhibitor (ITYP) were isolated from the culture filtrate of the pathogenic bacterium Yersinia pseudotuberculosis, and their biological activities were studied. YPTP was found to be highly toxic for random-bred white mice. Under in vitro conditions the proteolytic enzyme destroyed protective proteins of the immune system of the animals--IgG, IgA, and proteins of the complement system (CIq, C3, and C5)--and, consequently, was a pathogenetic factor in yersinioses. The inhibitor ITYP was shown to manifest antibacterial activity against virulent forms of Yersinia pseudotuberculosis, Escherichia coli, and Salmonella typhimurium. The ITYP preparation was harmless and nontoxic. PMID:10521713

  16. Intrinsic enzyme mimicking activity of gold nanoclusters upon visible light triggering and its application for colorimetric trypsin detection.

    PubMed

    Wang, Guang-Li; Jin, Lu-Yi; Dong, Yu-Ming; Wu, Xiu-Ming; Li, Zai-Jun

    2015-02-15

    In this research, a novel enzyme mimetics based on the photochemical property of gold nanoclusters was demonstrated. It was found that the bovine serum albumin (BSA) stabilized red or blue emitting gold nanoclusters (Au NCs) exhibited enzyme-like activity under visible light irradiation. The BSA-Au NCs had better stability against stringent conditions compared to natural enzyme. In addition, the photostimulated enzyme mimetics of BSA-Au NCs showed several unprecedented advantages over natural peroxidase or other existing alternatives based on nanomaterials, such as the independence of hydrogen peroxide on activity and the easily regulated activity by light irradiation. The mechanism of the photoresponsive enzyme-like activity of BSA-Au NCs was investigated. The photoactivated BSA-Au NCs was designed to develop a facile, cheap, and rapid colorimetric assay to detect trypsin through trypsin digestion of the protein template of BSA-stabilized Au NCs. The limit of detection for trypsin was 0.6 μg/mL, which was much lower than the average level of trypsin in patient's urine or serum. PMID:25310483

  17. Trypsin Isoinhibitors with Antiproliferative Activity toward Leukemia Cells from Phaseolus vulgaris cv “White Cloud Bean”

    PubMed Central

    Sun, Jian; Wang, Hexiang; Ng, Tzi Bun

    2010-01-01

    A purification protocol that comprised 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 was complied to isolate two trypsin inhibitors from Phaseolus vulgaris cv “White Cloud Bean”. Both trypsin inhibitors exhibited a molecular mass of 16 kDa and reduced the activity of trypsin with an IC50 value of about 0.6 μM. Dithiothreitol attenuated the trypsin inhibitory activity, signifying that an intact disulfide bond is indispensable to the activity. [Methyl-3H] thymidine incorporation by leukemia L1210 cells was inhibited with an IC50 value of 28.8 μM and 21.5 μM, respectively. They were lacking in activity toward lymphoma MBL2 cells and inhibitory effect on HIV-1 reverse transcriptase and fungal growth when tested up to 100 μM. PMID:20617140

  18. 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

    2014-01-01

    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. PMID:25317582

  19. 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. PMID:26769111

  20. Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

    PubMed Central

    Junker, Yvonne; Zeissig, Sebastian; Kim, Seong-Jun; Barisani, Donatella; Wieser, Herbert; Leffler, Daniel A.; Zevallos, Victor; Libermann, Towia A.; Dillon, Simon; Freitag, Tobias L.; Kelly, Ciaran P.

    2012-01-01

    Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4–MD2–CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients’ biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders. PMID:23209313

  1. Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables.

    PubMed

    Mosha, T C; Gaga, H E

    1999-01-01

    Proximate composition, energy, mineral and vitamin contents and the effect of blanching methods and times on the trypsin and chymotrypsin inhibitor activities were studied using cabbage, collard, turnip, peanut, and sweet potato leaves. Results of this study indicated that, crude protein, crude fat, carbohydrate and ash contents were in the range of 15.5-25.6%, 1.4-6.5%, 60.4-73.1% and 6.8-7.5%, respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%) while energy content per 100 g of vegetables was highest in sweet potato leaves (402 kcal) and lowest in cabbage (379 kcal). The mineral content per 100 g of vegetables were in the range of 33.4-249.8 mg, 241.2-471.2 mg, 12.1-75.1 mg, 14.9-98.9 mg, 0.5-3.5 mg and 0.9-3.1 mg for Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbic acid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of 45.1-112.7 mg, 0.2-0.3 mg, 0.3-0.8 mg and 2.0-7.3 mg, respectively. The trypsin inhibitory activity per gram of the vegetables was highest in collard (60.1 TIU/g) and lowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitor activity was highest in the peanut (69.6 CIU/g) but lowest in the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p < 0.05) reduced by most of the treatments in either the conventional or microwave blanching methods. In the conventional blanching method, trypsin inhibitor activity was reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsin inhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3 and 65.6 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. For the microwave oven blanching, trypsin inhibitor activity was reduced by 3.8, 3.3, 32.7, 5.0 and 9.5 percent

  2. Salmon and king crab trypsin stimulate interleukin-8 and matrix metalloproteinases via protease-activated receptor-2 in the skin keratinocytic HaCaT cell line.

    PubMed

    Bhagwat, Sampada S; Larsen, Anett K; Winberg, Jan-Olof; Seternes, Ole-Morten; Bang, Berit E

    2014-07-01

    Occupational skin symptoms are prevalent among the workers of the seafood processing industry. In this study we investigate the role of salmon (Salmo salar) and king crab trypsin (Paralithodes camtschaticus) as inducers of inflammation in skin via secretion of inflammatory mediators. Human skin keratinocytes (HaCaT cells) were exposed to purified salmon and king crab trypsin. We observed that salmon trypsin enhanced the secretion of IL-8 and MMP-2 and crab trypsin enhanced the secretion of IL-8, MMP-2 and MMP-9 in a dose dependent manner. As protease activated receptors (PAR)-2 in skin are known to play an important role in physiology and pathology, we explored the involvement of these receptors in mediating the release of interleukin (IL)-8 and matrix metalloproteinase (MMP)-2 and -9 subsequent to exposure of skin keratinocytes to salmon and crab trypsin. In addition we observed that salmon and crab trypsin exhibit individual differences in stimulating the release of these inflammatory mediators. Finally, using specific small interfering RNA (siRNA) against PAR-2, we confirmed that the increase in secretion of IL-8, MMP-2 and MMP-9 in skin keratinocytes following exposure to salmon and crab trypsin was mediated via activation of PAR-2. These results suggest that exposure to proteases from the seafood may lead to inflammatory reactions in skin. PMID:24795235

  3. Effect of mosquito midgut trypsin activity on dengue-2 virus infection and dissemination in Aedes aegypti.

    PubMed

    Molina-Cruz, Alvaro; Gupta, Lalita; Richardson, Jason; Bennett, Kristine; Black, William; Barillas-Mury, Carolina

    2005-05-01

    The effect of mosquito midgut trypsins in dengue serotype 2 flavivirus (DENV-2) infectivity to Aedes aegypti was studied. Addition of soybean trypsin inhibitor (STI) in a DENV-2 infectious blood meal resulted in a 91-97% decrease in midgut DENV-2 RNA copies (qRT-PCR analysis). STI treatment also resulted in slower DENV-2 replication in the midgut, less DENV-2 E protein expression, and decreased dissemination to the thorax and the head. A second uninfected blood meal, 7 days after the STI-treated infectious meal, significantly increased DENV-2 replication in the midgut and recovered oogenesis, suggesting that the lower viral infection caused by STI was in part due to a nutritional effect. Mosquitoes fed DENV-2 digested in vitro with bovine trypsin (before STI addition) exhibited a transient increase in midgut DENV-2 4 days postinfection. Blood digestion and possibly DENV-2 proteolytic processing, mediated by midgut trypsins, influence the rate of DENV-2 infection, replication, and dissemination in Ae. aegypti. PMID:15891140

  4. Roots of Contemporary Native American Activism. Commentary.

    ERIC Educational Resources Information Center

    Johnson, Troy R.

    1996-01-01

    Traces the foundations and development of Native American activism, 1950s-90s. Discusses relocation of reservation American Indians to urban areas in the 1950s without promised aid or vocational training, changing aspirations of Indian veterans and college students, lessons of the civil rights movement, occupations of Alcatraz Island and Wounded…

  5. 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

  6. 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

  7. Ammonia plasma treated polyethylene films for adsorption or covalent immobilization of trypsin: quantitative correlation between X-ray photoelectron spectroscopy data and enzyme activity.

    PubMed

    Ghasemi, Mahsa; Minier, Michel J G; Tatoulian, Michaël; Chehimi, Mohamed M; Arefi-Khonsari, Farzaneh

    2011-09-01

    The ammonia plasma process was used for generating reactive groups, particularly primary amine functions on the surface of polyethylene (PE) films, to immobilize the enzyme trypsin. The attachment of the enzyme was achieved by directly applying an aqueous solution of trypsin to the plasma-activated surface or by using glutaraldehyde as a chemical linker. In both cases, the utilization of sodium cyanoborohydride efficiently stabilized the immobilization. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and enzymatic activity measurements. Active trypsin was successfully immobilized on the surface with a mean activity of 0.09 ± 0.02 U/cm(2). The study of the stability of the immobilized enzyme during repetitive assays showed that some activity could be maintained during several months. An original quantitative correlation between the immobilized enzyme activity and the XPS signal intensity of the S 2p electrons present in the sulfur-containing amino acid residues was evidenced. PMID:21770448

  8. 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

  9. 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

    2016-01-01

    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

  10. Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin

    PubMed Central

    Sudom, Athena; Walters, Robert; Pastushok, Landon; Goldie, Douglas; Prasad, Lata; Delbaere, Louis T. J.; Goldie, Hughes

    2003-01-01

    The 1.8-Å resolution structure of the ATP-Mg2+-Ca2+-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg2+-Mn2+-pyruvate-PCK, except for the Ca2+ and Mn2+ binding sites. Ca2+ was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca2+ bound only at the active site, indicating that there is likely no surface allosteric site. 45Ca2+ bound to PCK with a Kd of 85 μM and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca2+. Separate roles of Mg2+, which binds the nucleotide, and Ca2+, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca2+-bound structure. Partial trypsin digestion abolishes Ca2+ activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca2+ binding site, probably stabilizing the C terminus. Phe409Ala, ΔPhe409, Phe413Ala, Δ397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca2+ site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes. PMID:12837799

  11. 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. PMID:25851516

  12. 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.

  13. Natural fermentation of lentils. Influence of time, concentration and temperature on protein content, trypsin inhibitor activity and phenolic compound content.

    PubMed

    Tabera, J; Frias, J; Estrella, I; Villa, R; Vidal-Valverde, C

    1995-12-01

    Lentil (Lens culinaris var. vulgaris) flour was naturally fermented for 4 days at different temperatures (28 degrees C, 35 degrees C and 42 degrees C) and concentrations (79 milligrams, 150 milligrams and 221 milligrams). Samples were analysed to establish the changes of total protein content and in vitro protein digestibility, trypsin inhibitor activity (TIA) and phenolic compound content during natural fermentation of lentils. The preparation of lentil flour suspensions to be fermented caused a slight increase in total protein and in vitro protein digestibility content, a decrease of TIA and a sharp decrease the tannin/catechin ratio. During the whole fermentation procedure, the minimum initial lentil concentration and temperature used (79 milligrams, 28 degrees C) achieved the maximum protein content and the lowest tannin/catechin ratio. The TIA was more affected by temperature than by concentration, and a 62.5% reduction was observed at 42 degrees C and 79 milligrams. PMID:8585337

  14. 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.

  15. Secretion of miraculin through the function of a signal peptide conserved in the Kunitz-type soybean trypsin inhibitor family.

    PubMed

    Takai, Ayako; Satoh, Makiko; Matsuyama, Tomomi; Ito, Akane; Nakata, Rieko; Aoyama, Takashi; Inoue, Hiroyasu

    2013-06-19

    Miraculin, a glycoprotein that modifies sour tastes into sweet ones, belongs to the Kunitz-type soybean trypsin inhibitor (STI) family. To clarify the functional relation of miraculin with Kunitz-type STIs, we investigated its subcellular localization and trypsin inhibitory activity. In transgenic Arabidopsis thaliana, miraculin, fused to yellow fluorescent protein, localized to and outside the plasma membrane depending on the putative secretion signal peptide. When transgenic seedlings were cultured in liquid medium, miraculin was present in the supernatant only after cellulase treatment. No trypsin inhibitory activity was detected in native or recombinant miraculin. In conclusion, miraculin is secreted outside the plasma membrane through the function of a signal peptide, conserved in Kunitz-type STIs, whereas its trypsin inhibitory activity may be lost during its evolution. PMID:23660404

  16. A preliminary neutron crystallography on the trypsin-bovine pancreatic trypsin inhibitor complex

    NASA Astrophysics Data System (ADS)

    Kawamura, K.; Yamada, T.; Kurihara, K.; Tamada, T.; Kuroki, R.; Tanaka, I.; Takahashi, H.; Niimura, N.

    2010-11-01

    Trypsin is one of serine proteases. BPTI (Bovine Pancreatic Trypsin Inhibitor) is a protein inhibitor, which binds trypsin tightly and inhibits cleavage of peptide bonds. X-ray structure determination of trypsin-BPTI complex could make clear the overview of the active site. However, information of hydrogen atoms related to catalytic mechanism has not been satisfied. In this study, the trypsin-BPTI complex structure has been determined by neutron diffraction data at 2.0 Å resolution. Deuterium atoms of catalytic triad, hydration structures in the binding pocket of trypsin and hydrogen bonds were observed. We would like to discuss details of hydrogen bonds in the interface between trypsin and BPTI and the adjacent water molecules including hydrogen atoms involved in the enzymatic reaction.

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

    PubMed Central

    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

    2011-01-01

    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 Taenia solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to E. granulosus Ag5 protein. The Taenia 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. PMID:21893105

  18. Elimination of trypsin inhibitor activity and beany flavor in soy milk by consecutive blanching and ultrahigh-temperature (UHT) processing.

    PubMed

    Yuan, Shaohong; Chang, Sam K C; Liu, Zhisheng; Xu, Baojun

    2008-09-10

    Soy foods contain significant health-promoting components but also may contain beany flavor and trypsin inhibitor activity (TIA), which can cause pancreatic disease if present at a high level. Thermal processing can inactivate TIA and lipoxygenase. Ultrahigh-temperature (UHT) processing is relatively new for manufacturing soy milk. Simultaneous elimination of TIA and soy odor by UHT processing for enhancing soy milk quality has not been reported. The objective was to determine TIA in soy milk processed by traditional, steam injection, blanching, and UHT methods and to compare the products with commercial soy milk products. Soybean was soaked and blanched at 70-85 degrees C for 30 s-7.5 min. The blanched beans were made into base soy milk. The hexanal content of the base soy milk was determined by gas chromatography to determine the best conditions for further thermal processing by indirect and direct UHT methods at 135-150 degrees C for 10-50 s using the Microthermics processor. Soy milk was also made from soaked soybeans by traditional batch cooking and steaming methods. Eighteen commercial products were selected from the supermarket. Residual TIA in soy milk processed by the traditional and steam injection to 100 degrees C for 20 min was approximately 13%. Blanching could inactivate 25-50% of TIAs of the raw soy milk. The blanch conditions of 80 degrees C and 2 min were selected for UHT processing because these conditions produced blanched soy milk without hexanal, indicating a complete heat inactivation of lipoxygenases. The TIA decreased with increased temperature and time of UHT heating. The maximal trypsin inhibitor inactivation was achieved by UHT direct and indirect methods with residual activities of approximately 10%. Some commercial soy milk products contained high TIAs. The results are important to the food industry and consumers. Kinetic analysis showed that heat inactivation (denaturation) of TIA, under the continuous processing conditions of the

  19. Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk.

    PubMed

    Atacan, Keziban; Çakıroğlu, Bekir; Özacar, Mahmut

    2016-12-01

    Trypsin (EC 3.4.21.4) was successfully immobilized on the surface of Fe3O4 magnetic nanoparticles that had been pre-treated with gallic acid (GA). Measurements of protein load by using Bradford assay and the trypsin-catalyzed hydrolysis of Nα-Benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) were made for the immobilized enzyme. By using magnetic nanoparticles, which provides easy separation and decent support material for enzyme immobilization with high surface area to volume ratio, and by employing biocompatible material gallic acid, immobilized enzyme system was synthesized along with improving trypsin activity and stability. Immobilized trypsin (TR) was more stable than the free one and demonstrated higher enzymatic activity at elevated temperatures (45-55°C) and in the alkaline pH region (6-10.5). Fe3O4 NPs-GA-TR retained 92% of its initial activity after 120days of storage at 4°C in sodium phosphate buffer (0.1M, pH 7.5), whereas the free trypsin maintained about 64% of its initial activity during the same storage period. In addition, activity of the immobilized trypsin was preserved 54.5% of its initial activity after eight times successive reuse. The Michaelis-Menten kinetic constant (Km) and maximum reaction velocity (Vmax) for free trypsin were 5.1mM and 23mM/min, respectively, whereas Km and Vmax values of immobilized trypsin were 7.88mM and 18.3mM/min, respectively. The performance of the immobilized trypsin was demonstrated by carrying out the hydrolysis of bovine serum albumin (BSA) within 1h, and the assay was performed by using liquid chromatography-mass spectrometry (LC-MS/MS) technique. The hydrolysis of bovine milk as a real food was investigated by immobilized trypsin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). PMID:27374556

  20. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies... 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...

  1. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies... 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...

  2. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies... 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...

  3. Sunflower trypsin inhibitor-1.

    PubMed

    Korsinczky, Michael L J; Schirra, Horst Joachim; Craik, David J

    2004-10-01

    SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors. PMID:15544530

  4. Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

    PubMed Central

    Weber, Michaela; Weber, Friedemann

    2014-01-01

    Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established. Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples. Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment. PMID:25146252

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Membrane affinity chromatography used for the separation of trypsin inhibitor.

    PubMed

    Guo, W; Shang, Z; Yu, Y; Guan, Y; Zhou, L

    1992-01-01

    Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability. PMID:1638098

  10. Active site titration of bovine beta-trypsin by N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis.

    PubMed

    Sartori, P; Djinovic Carugo, K; Ferraccioli, R; Balliano, G; Milla, P; Ascenzi, P; Bolognesi, M

    1995-01-16

    Kinetics of bovine beta-trypsin (trypsin) with the N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0 degrees C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin.N alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl.enzyme adduct has been solved by X-ray crystallography at 2.0 A resolution (R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue. PMID:7821429

  11. Haze activity of different barley trypsin inhibitors of the chloroform/methanol type (BTI-CMe).

    PubMed

    Ye, Lingzhen; Huang, Lu; Huang, Yuqing; Wu, Dezhi; Hu, Hongliang; Li, Chengdao; Zhang, Guoping

    2014-12-15

    Our previous study found that the critical protein in SE (silica eluted) proteins is BTI-CMe, and assumed that SE-ve malt for brewing may improve the haze stability in beer. In this study, we investigated the difference in gene sequence and corresponding amino acid sequence of BTI-CMe between SE+ve and SE-ve types. The results showed that there were 7 amino acid differences between Yerong (SE-ve) and Franklin (SE+ve). Two types BTI-CMe were expressed in vitro and purified successfully. By adding the purified BTI-CMe into commercial beer, we found that both original turbidity and alcohol chill haze degree of beer were increased. BTI-CMe of SE-ve haplotype showed a lower level of haze formation in beer than SE+ve haplotype. Response surface methodology (RSM) was conducted to determine the relationship between BTI-CMe and tannic acid, and their effects on haze formation. It was found that (1) higher content of BTI-CMe and/or tannic acid in beer would give rise to higher turbidity; (2) there was a significant interaction between BTI-CMe and tannic acid; (3) haze activity disparity of BTI-CMe between two types was significantly and positively correlated with the tannic acid concentration. PMID:25038664

  12. 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.

  13. Crystal structures of two engineered thiol trypsins.

    PubMed

    McGrath, M E; Wilke, M E; Higaki, J N; Craik, C S; Fletterick, R J

    1989-11-28

    We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 10(6)-fold loss of activity of trypsin S195C and the 10(8)-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2611228

  14. Trypsin and Chymotrypsin Test

    MedlinePlus

    ... trypsin/chymotrypsin test is used to help detect cystic fibrosis (CF) in symptomatic newborns and infants. Chymotrypsin may ... when a newborn or infant has symptoms of cystic fibrosis such as persistent diarrhea, foul-smelling bulky greasy ...

  15. 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.

  16. 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.

  17. 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.

  18. 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. PMID:26612727

  19. Isolation and characteristics of trypsin inhibitor from the hepatopancreas of a squid (Todarodes pacificus).

    PubMed

    Kishimura, H; Saeki, H; Hayashi, K

    2001-08-01

    Trypsin inhibitor was purified from the hepatopancreas of squid (Todarodes pacificus). The final inhibitor preparation was nearly homogeneous by SDS-PAGE with an estimated molecular weight of approximately 6300. The squid trypsin inhibitor was acid- and heat-stable, and active against trypsins from the pyloric ceca of starfish (Asterias amurensis) and saury (Cololabis saira) and porcine pancreatic trypsin. Amino acid composition of the squid trypsin inhibitor was compared with other invertebrate trypsin inhibitors. The squid trypsin inhibitor inhibited the autolysis of walleye pollock (Theragra chalcogramma) myofibrillar proteins. PMID:11470450

  20. Physical activity and Native Americans: a review.

    PubMed

    Coble, James D; Rhodes, Ryan E

    2006-07-01

    The physical activity behaviors of Native-American populations in the United States and Canada have received little attention in the health literature. The purpose of this review was to unite the literature regarding the physical activity behaviors of Native Americans. A majority of the literature was obtained using online databases. Reference lists were also reviewed to gain further access to the literature. Key-word searches included various combinations of Aboriginal, Native Indian, American Indian, Native American, First Nation, Métis, or Alaska Native with physical activity, exercise, and health behavior. Articles included were those published in English-language, peer-reviewed journals from 1990 until November 2005 that focused on participants aged 18 years and older. This review is organized according to ecologic models of health behavior, which take into account several correlates to explain human behavior, including demographic, personal health, environmental, and psychosocial. Correlates were included if they appeared at least three times in the literature. As a result of these inclusion criteria, the number of reviewed articles includes 28 quantitative, 4 qualitative, and 3 intervention studies. Results indicate that age, gender, and social support are important factors associated with physical activity. The remaining correlates show inconsistent or indeterminate results due in part to the paucity of research. It is suggested that an increase in the number of studies, especially those using longitudinal designs, is needed. Further, the application of psychosocial models to understand physical activity motivations as well as culturally appropriate and validated measurement tools are largely absent in the Native-American physical activity literature. PMID:16777541

  1. 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. PMID:27083826

  2. Antioxidant activities of trypsin inhibitor, a 33 KDa root storage protein of sweet potato (Ipomoea batatas (L.) Lam cv. Tainong 57).

    PubMed

    Hou, W C; Chen, Y C; Chen, H J; Lin, Y H; Yang, L L; Lee, M H

    2001-06-01

    Trypsin inhibitors (TIs), root storage proteins, were purified from sweet potato (Ipomoea batatas[L.] Lam cv. Tainong 57) roots by trypsin affinity column according to the methods of Hou and Lin (Plant Sci. 1997, 126, 11-19 and Plant Sci. 1997, 128, 151-158). A single band of 33 kDa TI was obtained by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This purified 33 kDa TI had scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. There was positive correlation between scavenging effects against DPPH (2 to 22%) and amounts of 33 kDa TI (1.92 to 46 pmol). The scavenging activities of 33 kDa TI against DPPH were calculated from linear regression to be about one-third of those of glutathione between 5 and 80 pmol. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detection, it was found that 33 kDa TI could capture hydroxyl radical, and the intensities of EPR signal were significantly decreased from 1.5 to 6 pmol of 33 kDa TI compared to those of the controls. It is suggested that 33 kDa TI, one of the sweet potato root storage proteins, may play a role as an antioxidant in roots and may be beneficial to health when it is consumed. PMID:11409996

  3. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  4. 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. PMID:26992797

  5. Using nonlinear ac electrokinetics vortex flow to enhance catalytic activities of sol-gel encapsulated trypsin in microfluidic devices

    PubMed Central

    Wang, Shau-Chun; Chen, Hsiao-Ping; Lai, Yi-Wen; Chau, Lai-Kwan; Chuang, Yu-Chun; Chen, Yi-Jie

    2007-01-01

    A novel microstirring strategy is applied to accelerate the digestion rate of the substrate Nα-benzoyl-L-arginine-4-nitroanilide (L-BAPA) catalyzed by sol-gel encapsulated trypsin. We use an ac nonlinear electrokinetic vortex flow to stir the solution in a microfluidic reaction chamber to reduce the diffusion length between the immobilized enzyme and substrate in the solution. High-intensity nonlinear electroosmotic microvortices, with angular speeds in excess of 1 cm∕s, are generated around a small (∼1.2 mm) conductive ion exchange granule when ac electric fields (133 V∕cm) are applied across a miniature chamber smaller than 10 μl. Coupling between these microvortices and the on-and-off electrophoretic motion of the granule in low frequency (0.1 Hz) ac fields produces chaotic stream lines to stir substrate molecules sufficiently. We demonstrate that, within a 5-min digestion period, the catalytic reaction rate of immobilized trypsin increases almost 30-fold with adequate reproducibility (15%) due to sufficient stirring action through the introduction of the nonlinear electrokinetic vortices. In contrast, low-frequency ac electroosmotic flow without the granule, provides limited stirring action and increases the reaction rate approximately ninefold with barely acceptable reproducibility (30%). Dye molecules are used to characterize the increases in solute diffusivity in the reaction reservoir in which sol-gel particles are placed, with and without the presence of granule, and compared with the static case. The solute diffusivity enhancement data show respective increases of ∼30 and ∼8 times, with and without the presence of granule. These numbers are consistent with the ratios of the enhanced reaction rate. PMID:19693360

  6. Neural activation in speech production and reading aloud in native and non-native languages.

    PubMed

    Berken, Jonathan A; Gracco, Vincent L; Chen, Jen-Kai; Soles, Jennika; Watkins, Kate E; Baum, Shari; Callahan, Megan; Klein, Denise

    2015-05-15

    We used fMRI to investigate neural activation in reading aloud in bilinguals differing in age of acquisition. Three groups were compared: French-English bilinguals who acquired two languages from birth (simultaneous), French-English bilinguals who learned their L2 after the age of 5 years (sequential), and English-speaking monolinguals. While the bilingual groups contrasted in age of acquisition, they were matched for language proficiency, although sequential bilinguals produced speech with a less native-like accent in their L2 than in their L1. Simultaneous bilinguals activated similar brain regions to an equivalent degree when reading in their two languages. In contrast, sequential bilinguals more strongly activated areas related to speech-motor control and orthographic to phonological mapping, the left inferior frontal gyrus, left premotor cortex, and left fusiform gyrus, when reading aloud in L2 compared to L1. In addition, the activity in these regions showed a significant positive correlation with age of acquisition. The results provide evidence for the engagement of overlapping neural substrates for processing two languages when acquired in native context from birth. However, it appears that the maturation of certain brain regions for both speech production and phonological encoding is limited by a sensitive period for L2 acquisition regardless of language proficiency. PMID:25776210

  7. 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

  8. [Biological activity of native and modified lipopolysaccharides of Pragia fontium].

    PubMed

    Varbanets', L D; Shubchyns'kyĭ, V V; Pokhyl, S I; Seĭfullina, I I; Shmatkova, N V; Samburs'kyĭ, S E

    2009-01-01

    Modified lipopolysaccharides (LPS) of Pragia fontium were obtained with germanium complexes (IV) of 2-aminobenzoylhydrazon of salicylic aldehyde (2-NH2-H2Bs), 2-hydroxybenzoylhydrazon salicylic aldehyde (2-OH-H2Bs) and nicotinoylhydrazon of salicylic aldehyde (H2Ns). The modification of LPS was confirmed by IR spectroscopy. Comparative investigations of pyrogenic activity of native and modified LPS showed, that only P. fontium 20125 LPS, modified by germanium complexes with 2-hydroxybenzoylhydrazon of salicylic aldehyde (2-OH-H2Bs) has lost the pyrogenic activity. In the homological reactions of double immunodiffusion in agar it was shown that all modified LPS unlike the native ones lose completely antigenic activity. PMID:19877414

  9. Inhibition of bovine beta-trypsin by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester: a kinetic and X-ray crystallographic study.

    PubMed

    Ascenzi, P; Balliano, G; Milla, P; Ferraccioli, R; Sartori, P; Djinovic-Carugo, K; Bolognesi, M

    1995-12-14

    Kinetics of the bovine beta-trypsin (trypsin) reaction with the active site titrant N alpha-(N,N-dimethylcarbamoyl)- alpha-aza-ornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) was obtained at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism of serine proteinases involving a stable acyl.enzyme adduct. Dmc-azaOrn-ONp binds stoichiometrically to trypsin and allows the reliable determination of the active enzyme concentration between 1.0 x 10(-6) M and 3.0 x 10(-4) M. The three-dimensional structure of the trypsin.Dmc-azaOrn acyl.enzyme adduct has been solved by X-ray crystallography at 1.8 A resolution (R = 0.153). The Dmc-azaOrn moiety of the active site titrant is accommodated in the serine proteinase active center, occupying the S1 specificity subsite, and is covalently linked to the OG atom of the Ser195 catalytic residue. PMID:7503719

  10. Dissociating Cortical Activity during Processing of Native and Non-Native Audiovisual Speech from Early to Late Infancy.

    PubMed

    Fava, Eswen; Hull, Rachel; Bortfeld, Heather

    2014-01-01

    Initially, infants are capable of discriminating phonetic contrasts across the world's languages. Starting between seven and ten months of age, they gradually lose this ability through a process of perceptual narrowing. Although traditionally investigated with isolated speech sounds, such narrowing occurs in a variety of perceptual domains (e.g., faces, visual speech). Thus far, tracking the developmental trajectory of this tuning process has been focused primarily on auditory speech alone, and generally using isolated sounds. But infants learn from speech produced by people talking to them, meaning they learn from a complex audiovisual signal. Here, we use near-infrared spectroscopy to measure blood concentration changes in the bilateral temporal cortices of infants in three different age groups: 3-to-6 months, 7-to-10 months, and 11-to-14-months. Critically, all three groups of infants were tested with continuous audiovisual speech in both their native and another, unfamiliar language. We found that at each age range, infants showed different patterns of cortical activity in response to the native and non-native stimuli. Infants in the youngest group showed bilateral cortical activity that was greater overall in response to non-native relative to native speech; the oldest group showed left lateralized activity in response to native relative to non-native speech. These results highlight perceptual tuning as a dynamic process that happens across modalities and at different levels of stimulus complexity. PMID:25116572

  11. 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

  12. Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L.).

    PubMed

    Sagili, Ramesh R; Pankiw, Tanya; Zhu-Salzman, Keyan

    2005-09-01

    Insecticidal properties of protease inhibitors have been established in transgenic plants. In the wake of continuous research and rapid development of protease inhibitors it is important to assess possible effects on beneficial insects like the honey bee (Apis mellifera L.). In this study, newly emerged caged bees were fed pollen diets containing three different concentrations (0.1%, 0.5% and 1% w:w) of soybean trypsin inhibitor (SBTI). Hypopharyngeal gland protein content, total midgut proteolytic enzyme activity of these bees, and survival were measured. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content and midgut proteolytic enzyme activity. There were no significant differences between control, 0.1% and 0.5% SBTI treatments. Bees fed a diet containing 1% SBTI had the lowest survival, followed by 0.5% and 0.1%, over a 30-day period. We concluded that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food, potentially threatening colony growth and maintenance. PMID:15927200

  13. Conformational activation of visual rhodopsin in native disc membranes.

    PubMed

    Malmerberg, Erik; M Bovee-Geurts, Petra H; Katona, Gergely; Deupi, Xavier; Arnlund, David; Wickstrand, Cecilia; Johansson, Linda C; Westenhoff, Sebastian; Nazarenko, Elena; Schertler, Gebhard F X; Menzel, Andreas; de Grip, Willem J; Neutze, Richard

    2015-03-10

    Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested. PMID:25759477

  14. 78 FR 32243 - Agency Information Collection Activities; Comment Request; Native American Career and Technical...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-29

    ... Agency Information Collection Activities; Comment Request; Native American Career and Technical Education... in response to this notice will be considered public records. Title of Collection: Native American... Annual Burden Hours: 1,200. Abstract: The Native American Career and Technical Education Program...

  15. Bulgur processes increase nutrition value: possible role in in-vitro protein digestability, phytic acid, trypsin inhibitor activity and mineral bioavailability.

    PubMed

    Ertaş, Nilgün; Türker, Selman

    2014-07-01

    Changes in the chemical constituents and nutritive quality of chickpea bulgur process, were studied in seeds that were soaked at different time (2, 8 and 12 h), different soaking water pH (pH 4, 6 and 8). Soaking in pH 8 soaking water and 12 h soaking time significantly (p < 0.05) reduced the ash content of chickpea bulgur samples. Compared to the raw material, the protein content and in-vitro protein digestibility increased, but starch, crude fiber, fat and energy values decreased and trypsin inhibitor activity was completely eliminated by bulgur process. As the soaking time increased, the phytic acid content also decreased. The highest total phenolic content was determinated with bulgur samples soaked in pH 4 soaking water. The P, Ca, and K values decreased with increasing soaking time. The HCl-extractability of P, Ca, Mg, Fe and K present in chickpea bulgur samples were significantly higher than the raw chickpea seeds. PMID:24966437

  16. 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.

  17. Interference by Gastrografin with a spectrophotometric trypsin assay

    PubMed Central

    Cowen, A. E.; McGeary, Heather M.; Campbell, C. B.

    1972-01-01

    Small quantities of Gastrografin remaining in the intestinal tract some hours after introduction have been shown to cause falsely low trypsin values as determined by a spectrophotometric assay system. This interference is due first to the high absorbance of Gastrografin at 254 nm resulting in a falsely high background optical density. Secondly, Gastrografin inhibits esterase activity towards the synthetic substrate used in this assay. Gastrografin did not interfere with gelatin proteolysis by trypsin and did not affect amylase or lipase determination. Thus the instillation of Gastrografin into the duodenum before pancreatic function tests should be avoided when the trypsin content is to be determined spectrophotometrically. PMID:5036096

  18. 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. PMID:15913299

  19. 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.

  20. 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.

  1. Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation.

    PubMed

    Gui, Yue; Qiu, Lingqi; Li, Yaohao; Li, Hongxing; Dong, Suwei

    2016-04-13

    Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1. PMID:26982082

  2. Sequence of three cDNAs encoding an alkaline midgut trypsin from Manduca sexta.

    PubMed

    Peterson, A M; Barillas-Mury, C V; Wells, M A

    1994-05-01

    We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta. PMID:8205142

  3. Investigation on potential enzyme toxicity of clenbuterol to trypsin

    NASA Astrophysics Data System (ADS)

    Chai, Jun; Xu, Qifei; Dai, Jinping; Liu, Rutao

    2013-03-01

    Clenbuterol (CLB) is a kind of β2-adrenergic agonists which was illegally used as feed additives nowadays. The toxic interaction of CLB with trypsin, an important digestive enzyme, was studied in vitro using multi-spectroscopic methods and molecular modeling methods. CLB was proved to bind with trypsin in S1 pocket, forming a complex driven by the dominant force of H-bond. The binding constant was calculated to be 1.79887 × 105 L mol-1 at 289 K and 0.32584 × 105 L mol-1 at 310 K, respectively. The skeleton of trypsin became loosened and unfolded with the amino residues microenvironment changed. The secondary and tertiary structure of trypsin also varied. Molecular modeling studies illustrated specific display of the binding information and explained most of the experiment phenomena. The binding site of CLB induced the fluorescence quenching as well as inhibition of enzyme activity of trypsin. The study confirmed that CLB had potential toxicity on both the structure and function of trypsin and the effects enhanced with the increasing concentration of CLB.

  4. 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.

  5. Engineering the primary substrate specificity of Streptomyces griseus trypsin.

    PubMed

    Page, Michael J; Wong, Sui-Lam; Hewitt, Jeff; Strynadka, Natalie C J; MacGillivray, Ross T A

    2003-08-01

    Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures. Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed. The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site. This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%). The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A). The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets. PMID:12885239

  6. 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…

  7. 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.

  8. Trypsin-Catalyzed Deltamethrin Degradation

    PubMed Central

    Xiong, Chunrong; Fang, Fujin; Chen, Lin; Yang, Qinggui; He, Ji; Zhou, Dan; Shen, Bo; Ma, Lei; Sun, Yan; Zhang, Donghui; Zhu, Changliang

    2014-01-01

    To explore if trypsin could catalyze the degradation of non-protein molecule deltamethrin, we compared in vitro hydrolytic reactions of deltamethrin in the presence and absence of trypsin with ultraviolet-visible (UV/Vis) spectrophotometry and gas chromatography-mass spectrometry (GC/MS). In addition, acute oral toxicity of the degradation products was determined in Wistar rats. The results show that the absorption peak of deltamethrin is around 264 nm, while the absorption peaks of deltamethrin degradation products are around 250 nm and 296 nm. In our GC setting, the retention time of undegraded deltamethrin was 37.968 min, while those of deltamethrin degradation products were 15.289 min and 18.730 min. The LD50 of deltamethrin in Wistar rats is 55 mg/kg, while that of deltamethrin degradation products is 3358 mg/kg in female rats and 1045 mg/kg in male rates (61-fold and 19-fold reductions in toxicity), suggesting that trypsin could directly degrade deltamethrin, which significantly reduces the toxicity of deltamethrin. These results expand people's understanding of the functions of proteases and point to potential applications of trypsin as an attractive agent to control residual pesticides in the environment and on agricultural products. PMID:24594869

  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. Purification and characterization of trypsins from the digestive tract of Locusta migratoria.

    PubMed

    Sakal, E; Applebaum, S W; Birk, Y

    1989-12-01

    Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site. PMID:2635697

  11. 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

  12. Multiple trypsin inhibitors from Momordica cochinchinensis seeds, the Chinese drug mubiezhi.

    PubMed

    Wong, Ricardo C H; Fong, W P; Ng, T B

    2004-02-01

    Five trypsin inhibitors, with N-terminal sequences demonstrating homology to each other and exhibiting a molecular weight of 5100, 4800, 4400, 4100, and 3900, respectively, were isolated from Momordica cochinchinensis seeds with a protocol involving acid extraction, ion exchange chromatography on SP-Sepharose chromatography, and RP-HPLC on a C18 column. Specific inhibitory activity against trypsin was demonstrated by the trypsin isoinhibitors with Ki values ranging from 5.3 x 10(-8) to 1.8 x 10(-6) M. None of the isoinhibitors could be cleaved by trypsin. PMID:15062996

  13. 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.

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

    SciTech Connect

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

    2009-04-01

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

  15. Mackerel Trypsin Purified from Defatted Viscera by Supercritical Carbon Dioxide

    PubMed Central

    Chun, Byung-Soo; Kishimura, Hideki; Nalinanon, Sitthipong; Klomklao, Sappasith; Benjakul, Soottawat

    2011-01-01

    Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and 60°C, respectively, using Nα-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above 40°C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO2 defatting process is useful as a substitute for organic solvent defatting process. PMID:22312468

  16. Dietary control of late trypsin gene transcription in Aedes aegypti.

    PubMed

    Noriega, F G; Barillas-Mury, C; Wells, M A

    1994-06-01

    In Aedes aegypti the levels of midgut trypsin activity after feeding are directly proportional to the protein concentration in the meal. The mechanisms of this up-regulatory event were investigated by analyzing the expression of the late trypsin gene under different dietary conditions. Transcription of the gene was dependent on both the quality and quantity of protein in the meal. As measured by Northern blot analysis, the levels of late trypsin gene expression increased up to 100-fold 24 h after feeding on gamma-globulin, hemoglobin or albumin (100 mg/ml). In contrast, gelatin, histone, amino acids, saline or agarose were very poor inducers of transcription. The rates of late trypsin transcription induced during the first 24 h were directly proportional to the concentration of protein in the meal. These data further support the suggestion that the primary mechanism that regulates the synthesis of trypsin in the mosquito midgut is transcriptional regulation of the gene. This regulatory mechanism enables the midgut to maintain the appropriate balance between protease synthesis and the protein content of the meal. PMID:7519098

  17. 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. PMID:22400467

  18. Trypsin-like Proteins of the Fungi as Possible Markers of Phytopathogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly available fungal genomes and related databases. Among the 74 fungal genomes, 30 species of parasitic Ascomycota contained...

  19. 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. PMID:27011349

  20. Antibody-mediated inhibition of Aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum.

    PubMed Central

    Shahabuddin, M; Lemos, F J; Kaslow, D C; Jacobs-Lorena, M

    1996-01-01

    The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission. PMID:8641775

  1. Spectroscopic investigations on the interactions between isopropanol and trypsin at molecular level

    NASA Astrophysics Data System (ADS)

    Hu, Xinxin; Yu, Zehua; Liu, Rutao

    2013-05-01

    The toxicity of hydroxyl group of isopropanol to trypsin in aqueous solution was investigated by techniques including UV-visible absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, enzyme activity assay and molecular docking technology. The results of UV-visible absorption spectroscopy and CD spectra indicate that isopropanol could change the secondary structure of trypsin by increasing the content of α-helix and decreasing the content of β-sheet. The tertiary structure of trypsin was also changed owing to the loss of environmental asymmetry of amino acid residues. Isopropanol bound into a hydrophobic cavity on the surface of trypsin by a hydrogen bond located between the hydrogen atom on the hydroxyl of isopropanol and the oxygen atoms on SER 214 and hydrophobic interaction, as the molecular docking results showed. In addition, isopropanol could affect the function of trypsin by increasing its catalytic activity.

  2. U.S. Geological Survey activities related to American Indians and Alaska Natives fiscal year 2004

    USGS Publications Warehouse

    U.S. Geological Survey; Brunstein, F. Craig, (Edited By)

    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.

  3. Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer.

    PubMed

    Roberts, C H; Chlebowski, J F

    1985-06-25

    The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects. PMID:3889000

  4. 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…

  5. 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.

  6. 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

  7. Modifications in trypsin digestion protocol for increasing the efficiency and coverage.

    PubMed

    Syal, Kirtimaan; Tadala, Raghu

    2015-01-01

    Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of 'staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information. PMID:25813035

  8. Mechanism of cinnamic acid-induced trypsin inhibition: A multi-technique approach

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    In order to investigate the association of the protease trypsin with cinnamic acid, the interaction was characterized by using fluorescence, UV-vis absorption spectroscopy, molecular modeling and an enzymatic inhibition assay. The binding process may be outlined as follows: cinnamic acid can interact with trypsin with one binding site to form cinnamic acid-trypsin complex, resulting in inhibition of trypsin activity; the spectroscopic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being -8.95 kJ mol-1 and 50.70 J mol-1 K-1, respectively. Noncovalent interactions make the main contribution to stabilize the trypsin-cinnamic acid complex; cinnamic acid can enter into the primary substrate-binding pocket and alter the environment around Trp and Tyr residues.

  9. The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain.

    PubMed Central

    Potempa, J; Pike, R; Travis, J

    1995-01-01

    Porphyromonas gingivalis contains high concentrations of numerous cysteine proteinases with trypsin-like activity which have been implicated as important virulence factors in adult-onset periodontitis. We have analyzed the subfractions of six P. gingivalis strains for the presence of arginine-X- and lysine-X-specific proteinases (Arg-gingipain [RGP] and Lys-gingipain [KGP]) previously purified from P. gingivalis H66. Western blot (immunoblot) analysis using antibodies produced against RGP and the N-terminal peptides of RGP or the catalytic subunit of KGP indicated that these enzymes are synthesized by the strains studied and exist as multiple molecular mass species. The major forms of RGP were identified as 110-, 95-, 70- to 90-, and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinins, with or without an added membrane anchorage peptide. The other forms are single-chain enzymes. While the 95- and 50-kDa RGP were found predominantly in culture medium, the 110- and 70- to 90-kDa forms associated with membranous fractions of the bacteria. The predominant form of KGP in all strains was a complex of the 60-kDa catalytic domain with hemagglutinins, and vesicle- and membrane-associated KGP was about 15 kDa larger than the 105-kDa enzyme present in culture media. These data explain the apparent complexity of P. gingivalis proteinases and indicate that in all strains tested there are two identical enzymes, one with arginine-X specificity and the other with lysine-X specificity, which, working in concert, are responsible for the trypsin-like activity associated with this bacterium. PMID:7890369

  10. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are....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...

  11. 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. PMID:27476144

  12. 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. PMID:27208744

  13. 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.

  14. 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. PMID:16161028

  15. 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…

  16. 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…

  17. 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…

  18. 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…

  19. An insulin with the native sequence but virtually no activity.

    PubMed

    Wollmer, A; Gilge, G; Brandenburg, D; Gattner, H G

    1994-03-01

    The B24-B25 peptide bond of insulin was replaced by an ester bond. To our knowledge this is the first replacement of a main chain atom reported for the hormone. It is meant to eliminate a structurally important H-bond between the imino group of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B chain from the underlying A chain. On the basis of independent experimental evidence this very conformational change is believed to be a prerequisite for receptor binding. It was thus anticipated that increased flexibility would increase receptor binding and activity. Intriguingly, porcine [B24-B25 CO-O]insulin (depsi-insulin) and likewise [B24-B25 CO-O]des-(B26-B30)insulin-B25-amide (depsi-DPI-amide) were found to be only 3-4% potent. PMID:8011179

  20. Chronic active thrombotic microangiopathy in native and transplanted kidneys.

    PubMed

    Zhang, Ping L; Prichard, Jeffery W; Lin, Fan; Shultz, Michael F; Malek, Sayeed K; Shaw, John H; Hartle, James E

    2006-01-01

    We report 2 complicated cases of thrombotic microangiopathy with chronic features and active components. The first case was a 36-yr-old woman with positive anti-DNA antibody and possible lupus cerebritis, who developed thrombotic microangiopathy secondary to a series of syndromes, including preeclampsia and anti-phospholipid antibody syndrome. Renal biopsy revealed no evidence of lupus nephritis and her renal function returned to normal 1 week after the biopsy. The second case was a 46-yr-old man who developed thrombotic microangiopathy of unknown etiology, which led to end-stage renal disease within 6 mo. The patient received a living related-donor transplant, but thrombotic microangiopathy recurred in the donor kidney only 40 days after the renal transplantation. PMID:16951274

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

    PubMed

    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

  2. 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

  3. Trypsin inactivation by latex fabricated gold nanoparticles: A new strategy towards insect control.

    PubMed

    Patil, Chandrashekhar D; Borase, Hemant P; Suryawanshi, Rahul K; Patil, Satish V

    2016-10-01

    Before applying nanotechnologies in biomedical and environmental areas it is advised to study interactions of nanoparticles and other nanomaterials with biomacromolecule present in living system. Moreover there is scarcity of reports on interactions between nanoparticles and biomaterials. In present report a rapid, ecofriendly method of fabricating stable gold nanoparticles (AuNPs) using latex of Jatropha curcas is reported for the first time. AuNPs found to have characteristic absorption maxima centered at 540nm, multiple irregular shapes with size range from 20 to 50nm and have crystalline nature. Latex fabricated AuNPs were found to inhibit catalytic potential of trypsin (a vital enzyme responsible for digestion, insecticide resistance and in several disease conditions). The interactions between AuNPs and trypsin were analyzed by UV-vis spectrophotometry and microwave plasma-atomic emission spectrometry which suggests formation of trypsin-AuNPs complex responsible for lowering catalytic activity of trypsin. Transmission electron microscopy, Fourier transform infrared spectroscopy and particle size distribution studies further confirm complex formation between trypsin and AuNPs. Diverse interactions of metal nanoparticles with proteins such as covalent interaction, electrostatic interactions and binding to SH group of amino acid may be the reasons behind inhibition of trypsin activity. In vivo studies on serum of several vectors and agriculturally important pests supported instrumental results on AuNPs induced trypsin inhibition. This work will bring a new research direction to explore eco-friendly nanoparticle in insect control via inhibition of enzyme catalytic potential. PMID:27542740

  4. Preparing bioactive surface of polystyrene with hydrophobin for trypsin immobilization

    NASA Astrophysics Data System (ADS)

    Niu, Baolong; Li, Bingzhang; Wang, Huifang; Guo, Ruijie; Liang, HaiXia; Qiao, Mingqiang; Li, Wenfeng

    2016-05-01

    A simple and reliable enzyme immobilization technique which can retain their catalytic activity for a long time is interest in many technologies. Here, the trypsin was immobilized by physisorption on polystyrene (PS) surface coated with a class I hydrophobin recombinant HGFI (rHGFI). X-ray photoelectron spectroscopy and water-contact-angle measurements demonstrated that the hydrophobicity of the PS could be well improved by rHGFI modification, and the self-assembled rHGFI showed an admirable stability on the hydrophobic PS surface against hot SDS rinsing. The enzyme activity assay illustrated that the capacity of rHGFI could enable it to well intermediate trypsin on PS surface and allow its immobilization lasting in an active form. The results obtained in this work show a way that surface modification with rHGFI should be an easy and feasible strategy for applications of enzyme-based catalytic surfaces in biosensing.

  5. Inhibition of trypsin expression in Lutzomyia longipalpis using RNAi enhances the survival of Leishmania

    PubMed Central

    2009-01-01

    Background Leishmania parasites must overcome several barriers to achieve transmission by their sand fly vectors. One of the earliest threats is exposure to enzymes during blood meal digestion. Trypsin-like enzymes appear to be detrimental to parasite survival during the very early phase of development as amastigotes transform into promastigote stages. Here, we investigate whether parasites can affect trypsin secretion by the sand fly midgut epithelium and if inhibition of this process is of survival value to the parasites. Results Infections of Lutzomyia longipalpis with Leishmania mexicana were studied and these showed that infected sand flies produced less trypsin-like enzyme activity during blood meal digestion when compared to uninfected controls. RNA interference was used to inhibit trypsin 1 gene expression by micro-injection into the thorax, as trypsin 1 is the major blood meal induced trypsin activity in the sand fly midgut. Injection of specific double stranded RNA reduced trypsin 1 expression as assessed by RT-PCR and enzyme assays, and also led to increased numbers of parasites in comparison with mock-injected controls. Injection by itself was observed to have an inhibitory effect on the level of infection, possibly through stimulation of a wound repair or immune response by the sand fly. Conclusion Leishmania mexicana was shown to be able to modulate trypsin secretion by Lutzomyia longipalpis to its own advantage, and direct inhibition of trypsin gene expression led to increased parasite numbers in the midguts of infected flies. Successful application of RNA interference methodology to Leishmania-infected sand flies now opens up the use of this technique to study a wide range of sand fly genes and their role in the parasite-vector interaction. PMID:20003192

  6. Measuring Physical Activity: Practical Approaches for Program Evaluation in Native American Communities

    PubMed Central

    Sallis, James F.

    2010-01-01

    Promoting physical activity is a high priority in the United States, especially for Native American populations, due to very high rates of inactivity-related chronic diseases. High quality physical activity measures can contribute to achieving health goals. Measuring a sample of the population can identify high risk subgroups and geographic locations that can be targeted for interventions. Outcomes of physical activity interventions should be evaluated because this is the only way to determine whether they are effective. Three types of measures are practical for use in non-research settings, though they still present challenges. First, self-reports are commonly used; they are low-cost, but the least accurate. Second, objective monitors such as pedometers, accelerometers, and heart rate monitors can provide accurate information, but resources and expertise are needed to collect and manage the data. Third, direct observation can be used to evaluate school physical education programs and assess how people are using parks and other physical activity facilities. Studies of Native American populations have used a variety of measures. Good evaluations can lead to program improvements, documenting positive results can attract funding to continue and expand programs, and communicating results can persuade other communities to adopt effective approaches. Program evaluations using quality physical activity measures can contribute to achieving the goal of improved health in Native American communities. PMID:20689389

  7. Measuring physical activity: practical approaches for program evaluation in Native American communities.

    PubMed

    Sallis, James F

    2010-01-01

    Promoting physical activity is a high priority in the United States, especially for Native American populations, due to very high rates of inactivity-related chronic diseases. High-quality physical activity measures can contribute to achieving health goals. Measuring a sample of the population can identify high-risk subgroups and geographic locations that can be targeted for interventions. Outcomes of physical activity interventions should be evaluated because this is the only way to determine whether they are effective. Three types of measures are practical for use in nonresearch settings, although they still present challenges. First, self-reports are commonly used; they are low-cost but the least accurate. Second, objective monitors such as pedometers, accelerometers, and heart rate monitors can provide accurate information, but resources and expertise are needed to collect and manage data. Third, direct observation can be used to evaluate school physical education programs and assess how people are using parks and other physical activity facilities. Studies of Native American populations have used a variety of measures. Good evaluations can lead to program improvements, documenting positive results can attract funding to continue and expand programs, and communicating results can persuade other communities to adopt effective approaches. Program evaluations using quality physical activity measures can contribute to achieving the goal of improved health in Native American communities. PMID:20689389

  8. Conjugation of D-glucosamine to bovine trypsin increases thermal stability and alters functional properties.

    PubMed

    Gizurarson, Jóhann Grétar Kröyer; Filippusson, Hörður

    2015-01-01

    D-Glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 D-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca(2+) ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k(cat)/K(m)) than unmodified trypsin against the substrate N-α-benzoyl-L-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate. PMID:26047909

  9. 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

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

    PubMed

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

    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

  11. Reduced bovine pancreatic trypsin inhibitor has a compact structure.

    PubMed

    Amir, D; Haas, E

    1988-12-13

    The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions. PMID:2466482

  12. Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

    PubMed

    Krishna Das, Bhaba; Kumar, Amit; Maindola, Priyank; Mahanty, Srikrishna; Jain, S K; Reddy, Mallireddy K; Arockiasamy, Arulandu

    2016-05-13

    Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. PMID:27067046

  13. Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin.

    PubMed

    Blanco, R M; Alvaro, G; Guisán, J M

    1991-07-01

    By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst. PMID:1367640

  14. Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera.

    PubMed

    Kishimura, Hideki; Hayashi, Kenji

    2002-06-01

    Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found. PMID:12031475

  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. 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.

  17. Interaction of Cu(2+), Pb (2+), Zn (2+) with trypsin: what is the key factor of their toxicity?

    PubMed

    Zhang, Tong; Zhang, Hao; Liu, Guiliang; Gao, Canzhu; Liu, Rutao

    2014-11-01

    Heavy metals possess great endangerment to environment even human health because of their indissolubility and bioaccumulation. The toxicity of heavy metal ions (Cu(2+), Pb(2+), Zn(2+)) to trypsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and enzyme activity assay. The experimental results showed that toxic effect of heavy metal ions was due to their own characteristic, rather than the electric charges of the ion. Zn(2+) could not show the obvious toxicity to trypsin, while the structure and function of trypsin was damaged when the enzyme explored to Cu(2+) and Pb(2+). From the spectra results, we found that Cu(2+) would bind with trypsin, which lead to the fluorescence quenched and hydrophobicity increased. Pb(2+) could also change the structure and reduce the activity of trypsin in high concentration. In vitro measurement, the toxicity order of heavy metal ions to trypsin is: Cu(2+) > Pb(2+) > Zn(2+). In addition, isothermal titration calorimetry analysis proved that the interactions between Cu(2+), Pb(2+), Zn(2+) and trypsin were all spontaneous and exothermic, which indicated the adverse effect of these heavy metal ions to trypsin. PMID:25323557

  18. Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling.

    PubMed

    Song, Wei; Yu, Zehua; Hu, Xinxin; Liu, Rutao

    2015-02-25

    Studies on the effects of environmental pollutants to protein in vitro has become a global attention. Hydrogen peroxide (H2O2) is used as an effective food preservative and bleacher in industrial production. The toxicity of H2O2 to trypsin was investigated by multiple spectroscopic techniques and the molecular docking method at the molecular level. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the trypsin-H2O2 complex on account of the negative ΔH(0) and ΔS(0). The binding of H2O2 changed the conformational structures and internal microenvironment of trypsin illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) results. However, the binding site was far away from the active site of trypsin and the trypsin activity was only slightly affected by H2O2, which was further explained by molecular docking investigations. PMID:25228036

  19. Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling

    NASA Astrophysics Data System (ADS)

    Song, Wei; Yu, Zehua; Hu, Xinxin; Liu, Rutao

    2015-02-01

    Studies on the effects of environmental pollutants to protein in vitro has become a global attention. Hydrogen peroxide (H2O2) is used as an effective food preservative and bleacher in industrial production. The toxicity of H2O2 to trypsin was investigated by multiple spectroscopic techniques and the molecular docking method at the molecular level. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the trypsin-H2O2 complex on account of the negative ΔH0 and ΔS0. The binding of H2O2 changed the conformational structures and internal microenvironment of trypsin illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) results. However, the binding site was far away from the active site of trypsin and the trypsin activity was only slightly affected by H2O2, which was further explained by molecular docking investigations.

  20. 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

  1. 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

    2016-01-01

    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

  2. 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

  3. 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. PMID:27109756

  4. The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis.

    PubMed

    Egeland, Siri Valen; Reubsaet, Léon; Halvorsen, Trine Grønhaug

    2016-05-10

    The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60°C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times <4h. The accelerated digestion protocol (1:1 (w/w), 45min) was applied to realistic human serum samples containing the biomarker protein hCG to demonstrate its applicability. PMID:26907699

  5. Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea.

    PubMed

    Castillo-Yáñez, Francisco Javier; Pacheco-Aguilar, Ramón; García-Carreño, Fernando Luis; Navarrete-Del Toro, María de Los Angeles

    2005-01-01

    Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica. PMID:15621514

  6. 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.

  7. 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

  8. Human β-Defensin 4 with Non-Native Disulfide Bridges Exhibit Antimicrobial Activity

    PubMed Central

    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. PMID:25785690

  9. Modulation of chymase-mediated rat serosal mast cell degranulation by trypsin or diisopropyl fluorophosphate.

    PubMed

    Schick, B; Austen, K F

    1989-03-01

    Exposure of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, at 37 degrees results in exocytosis, as determined by beta-hexosaminidase release. As the number of RSMC is increased with a set amount of chymase, the net percentage beta-hexosaminidase release decreases linearly, implying a finite set of cellular interactions per chymase unit. Pretreatment of RSMC with trypsin at 37 degrees renders them refractory to subsequent exocytosis mediated by chymase in a dose- and time-dependent fashion, with complete refractiveness occurring by 15 min at 37 degrees with 2.5 micrograms trypsin/ml. Anti-IgE-mediated coupled activation-secretion of RSMC is not affected by the same trypsin pretreatment. When RSMC are pretreated with trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree a progressive loss of sensitivity to activation by chymase at 37 degrees occurs. RSMC susceptibility to chymase-mediated degranulation after trypsin pretreatment can be partially regenerated by culturing the RSMC for about 24 hr in medium at 37 degrees. These findings suggest that a trypsin-sensitive constituent, possibly a receptor or substrate, is necessary for the functional interaction of chymase with RSMC. When added with diisopropyl fluorophosphate (DFP), chymase does not induce RSMC degranulation at 37 degrees. However, if the DFP is removed before addition of chymase at 37 degrees or is added after the chymase-priming event occurs at 1 degree, subsequent degranulation at 37 degrees is not inhibited. Thus, the induction and not the secretion phase is DFP-inhibitable in chymase-induced activation-secretion. In addition, the priming but not the exocytosis phase of chymase-initiated RSMC activation-secretion, which is not dependent on temperature and calcium ion concentration, involves a cellular trypsin-sensitive protein. PMID:2522909

  10. Modulation of chymase-mediated rat serosal mast cell degranulation by trypsin or diisopropyl fluorophosphate.

    PubMed Central

    Schick, B; Austen, K F

    1989-01-01

    Exposure of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, at 37 degrees results in exocytosis, as determined by beta-hexosaminidase release. As the number of RSMC is increased with a set amount of chymase, the net percentage beta-hexosaminidase release decreases linearly, implying a finite set of cellular interactions per chymase unit. Pretreatment of RSMC with trypsin at 37 degrees renders them refractory to subsequent exocytosis mediated by chymase in a dose- and time-dependent fashion, with complete refractiveness occurring by 15 min at 37 degrees with 2.5 micrograms trypsin/ml. Anti-IgE-mediated coupled activation-secretion of RSMC is not affected by the same trypsin pretreatment. When RSMC are pretreated with trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree a progressive loss of sensitivity to activation by chymase at 37 degrees occurs. RSMC susceptibility to chymase-mediated degranulation after trypsin pretreatment can be partially regenerated by culturing the RSMC for about 24 hr in medium at 37 degrees. These findings suggest that a trypsin-sensitive constituent, possibly a receptor or substrate, is necessary for the functional interaction of chymase with RSMC. When added with diisopropyl fluorophosphate (DFP), chymase does not induce RSMC degranulation at 37 degrees. However, if the DFP is removed before addition of chymase at 37 degrees or is added after the chymase-priming event occurs at 1 degree, subsequent degranulation at 37 degrees is not inhibited. Thus, the induction and not the secretion phase is DFP-inhibitable in chymase-induced activation-secretion. In addition, the priming but not the exocytosis phase of chymase-initiated RSMC activation-secretion, which is not dependent on temperature and calcium ion concentration, involves a cellular trypsin-sensitive protein. PMID:2522909

  11. Organogenesis of exocrine pancreas in sharpsnout sea bream (Diplodus puntazzo) larvae: characterization of trypsin expression.

    PubMed

    Kamaci, H Okan; Suzer, Cüneyt; Coban, Deniz; Saka, Sahin; Firat, Kürşat

    2010-12-01

    The ontogeny and differentiation stages of digestive systems related with trypsin expression in larvae of sharpsnout sea bream, Diplodus puntazzo, were investigated from hatching to 40 DAH (days after hatching), and total lengths and weights of larvae were determined. Histologic and enzymatic techniques were used to explain the functional development of the pancreas including trypsin activity. The pancreas was identified as a compact structure located in the region slightly posterior to the liver. At 3 DAH, first anus and then mouth opened. Incipient pancreas secretion polyhedral cells could be first observed as zymogen granules. During larval metamorphosis, the pancreas became diffuse, spreading throughout the mesentery in proximity to the stomach, the anterior intestine and the pyloric caeca. The specific activity of trypsin (42.54±6.8 mU/mg protein(-1)) was found as early as after hatching at larvae size of 2.87±0.34 mm at 0 DAH. Activity further increased until 10 DAH, especially after exogenous feeding. The highest trypsin activity was detected at 25 DAH as 119.26±11.6 mU/mg protein(-1). It is concluded that exocrine pancreas organogenesis is the main critical step in the development of digestive system that results in zymogen granules accumulation and increased trypsin activity. PMID:20077135

  12. 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

  13. Sources of antioxidant activity in Australian native fruits. Identification and quantification of anthocyanins.

    PubMed

    Netzel, Michael; Netzel, Gabriele; Tian, Qingguo; Schwartz, Steven; Konczak, Izabela

    2006-12-27

    Selected native Australian fruits, muntries (Kunzea pomifera F. Muell., Myrtaceae), Tasmanian pepper berry (Tasmanian lanceolata R. Br., Winteraceae), Illawarra plum (Podocarpus elatus R. Br. ex Endl., Podocarpaceae), Burdekin plum (Pleiogynium timorense DC. Leenh, Anacardiaceae), Cedar Bay cherry (Eugenia carissoides F. Muell., Myrtaceae), Davidson's plum (Davidsonia pruriens F. Muell. var. pruriens, Davidsoniaceae), and Molucca raspberry (Rubus moluccanus var. austropacificus van Royen, Rosaceae), were evaluated as sources of antioxidants by 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays and compared with blueberry (Vaccinum spp. cv. Biloxi). The total reducing capacity of five fruits was 3.5-5.4-fold higher than that of blueberry, and the radical scavenging activities of muntries and Burdekin plum were 1.5- and 2.6-fold higher, respectively. The total phenolic level by Folin-Ciocalteu assay highly correlated with the antioxidant activity. Therefore, systematic research was undertaken to identify and characterize phenolic complexes. In the present study we report on the levels and composition of anthocyanins. The HPLC-DAD and HPLC/ESI-MS-MS (ESI = electrospray ionization) analyses revealed simple anthocyanin profiles of one to four individual pigments, with cyanidin as the dominating type. This is the first evaluation of selected native Australian fruits aiming at their utilization for the development of novel functional food products. PMID:17177507

  14. 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

  15. Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor*

    PubMed Central

    Daly, Norelle L.; Thorstholm, Louise; Greenwood, Kathryn P.; King, Gordon J.; Rosengren, K. Johan; Heras, Begoña; Martin, Jennifer L.; Craik, David J.

    2013-01-01

    MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. PMID:24169696

  16. Native and recombinant Pg-AMP1 show different antibacterial activity spectrum but similar folding behavior.

    PubMed

    Porto, William F; Nolasco, Diego O; Franco, Octavio L

    2014-05-01

    Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and they can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both native and recombinant peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide. PMID:24582624

  17. Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

    PubMed Central

    Boschwitz, H; Milner, Y; Keynan, A; Halvorson, H O; Troll, W

    1983-01-01

    The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T. PMID:6401704

  18. Transcriptional Control by A-Factor of Two Trypsin Genes in Streptomyces griseus

    PubMed Central

    Kato, Jun-ya; Chi, Won-Jae; Ohnishi, Yasuo; Hong, Soon-Kwang; Horinouchi, Sueharu

    2005-01-01

    AdpA is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus, forming an AdpA regulon. Trypsin-like activity was detected at a late stage of growth in the wild-type strain but not in an A-factor-deficient mutant. Consistent with these observations, two trypsin genes, sprT and sprU, in S. griseus were found to be members of the AdpA regulon; AdpA activated the transcription of both genes by binding to the operators located at about −50 nucleotide positions with respect to the transcriptional start point. The transcription of sprT and sprU, induced by AdpA, was most active at the onset of sporulation. Most trypsin activity exerted by S. griseus was attributed to SprT, because trypsin activity in an sprT-disrupted mutant was greatly reduced but that in an sprU-disrupted mutant was only slightly reduced. This was consistent with the observation that the amount of the sprT mRNA was much greater than that of the sprU transcript. Disruption of both sprT and sprU (mutant ΔsprTU) reduced trypsin activity to almost zero, indicating that no trypsin genes other than these two were present in S. griseus. Even the double mutant ΔsprTU grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain. PMID:15601713

  19. A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity-Based Probes: Diubiquitin as a Case Study

    PubMed Central

    Mulder, Monique P C; El Oualid, Farid; ter Beek, Jarno; Ovaa, Huib

    2014-01-01

    We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity-based probes based on all seven isopeptide-linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage-specific manner. PMID:24623714

  20. 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. PMID:23017386

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

    PubMed

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

    2006-02-01

    Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively. PMID:16406091

  2. 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

  3. The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin.

    PubMed

    Komuro, T; Miwa, S; Minowa, T; Okamoto, Y; Enoki, T; Ninomiya, H; Zhang, X F; Uemura, Y; Kikuchi, H; Masaki, T

    1997-03-01

    1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca2+ concentrations ([Ca2+]i) in dispersed rabbit vascular smooth muscle cells with a Ca2+ indicator fura-2. The actions of trypsin were compared with those of thrombin. 2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. 3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin. 4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca2+, indicating that most of the contraction is dependent on extracellular Ca2+. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca2+ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca2+ channels are involved in these contractions. 5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it's protease activity. 6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca2+]i, but an application of

  4. 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

  5. Antioxidant and antimycotic activities of two native lavandula species from portugal.

    PubMed

    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

  6. The effect of thermal treatment of whole soybean flour on the conversion of isoflavones and inactivation of trypsin inhibitors.

    PubMed

    Andrade, J C; Mandarino, J M G; Kurozawa, L E; Ida, E I

    2016-03-01

    The aim of this paper was to evaluate the effect of the thermal treatment of whole soybean flour (WSF) on the conversion of isoflavones and the inactivation of trypsin inhibitors. Soybeans were ground and whole soybean flour was obtained and subjected to heat treatment in an oven for 10, 15 and 20min at 100, 150 and 200°C according to a 3(2) experimental design. The response functions were taken to be the contents of different isoflavone forms and the residual activity of trypsin inhibitors. The thermal treatment in the oven altered the content and profile of the different isoflavones forms. At 200°C for 20min, there was a higher conversion of malonylglycosides to acetylglycosides, β-glycosides and aglycones and a significant reduction in the activity of trypsin inhibitors. Mathematical models were established to estimate the process parameters in obtaining the WSF with isoflavone conversions and reductions in trypsin inhibitor activity. PMID:26471658

  7. Larvicidal and cytotoxic activities of extracts from 11 native plants from northeastern Mexico.

    PubMed

    de la Torre Rodriguez, Yael C; Martínez Estrada, Francisco Ricardo; Flores Suarez, Adriana Elizabeth; Waksman de Torres, Noemí; Salazar Aranda, Ricardo

    2013-03-01

    Of all mosquito-borne viral diseases, dengue is spreading most rapidly worldwide. Conventional chemical insecticides (e.g., organophosphates and carbamates) effectively kill mosquitoes at their larval stage, but are toxic to humans. Natural product-based insecticides may be highly specific. Herein, we report the insecticidal activities of 11 native Mexican plants against Aedes aegypti (L). Ether extracts of Ambrosia confertiflora De Candolle, Thymus vulgaris (L.), and Zanthoxylum fagara (L.), and both ether and methanol extracts of Ruta chalepensis L. were significantly larvicidal toward the dengue mosquito after 24 h of exposure. Of them, only the ether extract of A. confertiflora was toxic to Vero cells. In conclusion, the ether extracts of Thymus vulgaris, Z. fagara, and both ether and methanol extracts of Ruta chalepensis L., could be considered as potential bioinsecticides. PMID:23540118

  8. 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. PMID:24338707

  9. 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.

  10. Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A

    PubMed Central

    Zarei, Mandana; Aminzadeh, Saeed; Zolgharnein, Hossein; Safahieh, Alireza; Daliri, Morteza; Noghabi, Kambiz Akbari; Ghoroghi, Ahmad; Motallebi, Abbasali

    2011-01-01

    Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. In the following investigation, a novel chitinase with antifungal activity was characterized from a native Serratia marcescens B4A. Partially purified enzyme had an apparent molecular mass of 54 kDa. It indicated an optimum activity in pH 5 at 45°C. Enzyme was stable in 55°C for 20 min and at a pH range of 3–9 for 90 min at 25°C. When the temperature was raised to 60°C, it might affect the structure of enzymes lead to reduction of chitinase activity. Moreover, the Km and Vmax values for chitin were 8.3 mg/ml and 2.4 mmol/min, respectively. Additionally, the effect of some cations and chemical compounds were found to stimulate the chitinase activity. In addition, Iodoacetamide and Idoacetic acid did not inhibit enzyme activity, indicating that cysteine residues are not part of the catalytic site of chitinase. Finally, chitinase activity was further monitored by scanning electronic microscopy data in which progressive changes in chitin porosity appeared upon treatment with chitinase. This enzyme exhibited antifungal activity against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola, revealing a potential application for the industry with potentially exploitable significance. Fungal chitin shows some special features, in particular with respect to chemical structure. Difference in chitinolytic ability must result from the subsite structure in the enzyme binding cleft. This implies that why the enzyme didn’t have significant antifungal activity against other Fungi. PMID:24031719

  11. The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis.

    PubMed

    Mailloux, Ryan J; Darwich, Rami; Lemire, Joseph; Appanna, Vasu

    2008-04-01

    Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival. PMID:18324728

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

    PubMed

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

    2016-05-01

    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

  13. 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

  14. 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

  15. 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. PMID:26613511

  16. 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.

  17. Investigate the binding of catechins to trypsin using docking and molecular dynamics simulation.

    PubMed

    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

  18. 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 ...

  19. Growth Impairment Caused by Raw Linseed Consumption: Can Trypsin Inhibitors Be Harmful for Health?

    PubMed

    Anaya, Katya; Cruz, Ana C B; Cunha, Dayse C S; Monteiro, Sandra M N; Dos Santos, Elizeu A

    2015-09-01

    Linseed (Linun usitatissimum L.) is an important oilseed whose nutritional value can be impaired due to presence of antinutritional factors and low protein digestibility. Protein fractions from raw linseed meal were extracted, isolated and analyzed in vitro and in vivo. Globulins, the major protein fraction of linseed, showed low in vitro susceptibility to trypsin and chymotrypsin, but its in vivo digestibility was 93.2 %. Albumin fraction had high trypsin inhibition activity (5250 Inhibition Units g(-1)) and presented low molecular mass protein bands, similar to known trypsin inhibitors. Raw linseed consumption caused negative effects on rat growth and reduction of intestinal villi. Results indicate that raw linseed meal must not be used as an exclusive source of protein regardless of the major proteins have high digestibility; digestive enzymes inhibitors in raw linseed probably reduces the protein utilization. PMID:26243664

  20. 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.

  1. 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

  2. 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…

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

    PubMed

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

    2006-06-01

    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. PMID:16734450

  4. PCB Exposure and in Vivo CYP1A2 Activity among Native Americans

    PubMed Central

    Fitzgerald, Edward F.; Hwang, Syni-An; Lambert, George; Gomez, Marta; Tarbell, Alice

    2005-01-01

    Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects. PMID:15743714

  5. 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...

  6. Polyphenols and antioxidant activity of calafate ( Berberis microphylla ) fruits and other native berries from Southern Chile.

    PubMed

    Ruiz, Antonieta; Hermosín-Gutiérrez, Isidro; Mardones, Claudia; Vergara, Carola; Herlitz, Erika; Vega, Mario; Dorau, Carolin; Winterhalter, Peter; von Baer, Dietrich

    2010-05-26

    Calafate ( Berberis microphylla ) is a native berry grown in the Patagonian area of Chile and Argentina. In the present study the phenolic composition and antioxidant activity of its fruits were studied and also compared with data obtained for other berry fruits from southern Chile including maqui ( Aristotelia chilensis ) and murtilla ( Ugni molinae ). Polyphenolic compounds in calafate fruit were essentially present in glycosylated form, 3-glucoside conjugates being the most abundant anthocyanins. The anthocyanin content in calafate berries (17.81 +/- 0.98 micromol g(-1)) and flavonol level (0.16 +/- 0.01 micromol g(-1)) are comparable with those found in maqui (17.88 +/- 1.15 and 0.12 +/- 0.01 micromol g(-1), respectively); however, maqui shows lower flavan-3-ol concentration than calafate (0.11 +/- 0.01 and 0.24 +/- 0.03 micromol g(-1), respectively). Maqui and calafate show high antioxidant activity, which correlates highly with total polyphenol content and with anthocyanin concentration. PMID:20438111

  7. 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. PMID:26830583

  8. Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter

    PubMed Central

    Bodnar, Anastasia L.; Schroder, Megan N.; Scott, M. Paul

    2016-01-01

    High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected control populations. These higher levels of the 27kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression. PMID:26895451

  9. 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

  10. New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

    PubMed Central

    Rodríguez, Javier M.; Chichón, Francisco J.; Martín-Forero, Esther; González-Camacho, Fernando; Carrascosa, José L.; Castón, José R.; Luque, Daniel

    2014-01-01

    The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry. PMID:24873828

  11. A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting.

    PubMed

    Ibelli, Adriana M G; Kim, Tae K; Hill, Creston C; Lewis, Lauren A; Bakshi, Mariam; Miller, Stephanie; Porter, Lindsay; Mulenga, Albert

    2014-05-01

    Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host's defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host's anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host's hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick-host interface. PMID:24583183

  12. Presenilin 1 promotes trypsin-induced neuroprotection via the PAR2/ERK signaling pathway. Effects of presenilin 1 FAD mutations.

    PubMed

    Nikolakopoulou, Angeliki M; Georgakopoulos, Anastasios; Robakis, Nikolaos K

    2016-06-01

    Mutants of presenilin 1 (PS1) increase neuronal cell death causing autosomal-dominant familial Alzheimer's disease (FAD). Recent literature shows that treatment of neuronal cultures with low concentrations of trypsin, a member of the serine family of proteases, protects neurons from toxic insults by binding to the proteinase-activated receptor 2 and stimulating survival kinase extracellular signal-regulated kinase (ERK 1/2). Other studies show that PS1 is necessary for the neuroprotective activity of specific neurotrophic factors, such as brain-derived neurotrophic factor, against excitotoxicity and oxidative stress. Here, we show that treatment of mouse cortical neuronal cultures with trypsin activates ERK1/2 and protects neurons against glutamate excitoxicity. The trypsin-dependent ERK activation and neuroprotection requires both alleles of PS1 because neither PS1 knockout nor PS1 hemizygous neuronal cultures can use exogenous trypsin to activate ERK1/2 or increase neuronal survival. The protective effect of PS1 does not depend on its γ-secretase activity because inhibitors of γ-secretase have no effect on trypsin-mediated neuroprotection. Importantly, cortical neuronal cultures either heterozygous or homozygous for PS1 FAD mutants are unable to use trypsin to activate ERK1/2 and rescue neurons from excitotoxicity, indicating that FAD mutants inhibit trypsin-dependent neuroprotection in an autosomal-dominant manner. Furthermore, our data support the theory that PS FAD mutants increase neurodegeneration by inhibiting the ability of neurons to use cellular factors as protective agents against toxic insults. PMID:27143420

  13. Trypsin from the digestive system of carp Cirrhinus mrigala: purification, characterization and its potential application.

    PubMed

    Khangembam, Bronson Kumar; Chakrabarti, Rina

    2015-05-15

    Trypsin was purified 35.64-fold with 4.97% recovery from the viscera of carp Cirrhinus mrigala (mrigal) by ammonium sulfate precipitation, ion exchange and affinity chromatography. The purified enzyme was active at a wide range of pH (7.0-9.2) and temperature (10-50°C). The purified enzyme exhibited high thermal stability up to 50°C for 1h. The enzyme activity was stabilized by Ca(+2) (2mM) up to 7h at 40°C. The Km and kcat values of purified enzyme were 0.0672 mM and 92.09/s/mM, respectively. Soybean trypsin inhibitor and phenylmethylsulphonylflouride completely inhibited the enzyme activity. The specific inhibitor of trypsin, N-α-p-tosyl-L-lysine chloromethyl ketone inhibited 99.67% activity. Na(+), K(+) and Li(+) inhibited 20.99 ± 5.25%, 16.53 ± 4.80% and 18.99 ± 1.42% of enzyme activity, respectively. Divalent ions Mg(+2), Zn(+2), Co(+2), Hg(+2) and Cd(+2) inhibited 21.61 ± 2.22%, 31.62 ± 1.78%, 31.62 ± 1.96%, 85.68 ± 1.51% and 47.95 ± 2.13% enzyme activity, respectively. SDS-PAGE showed that the molecular mass of purified enzyme was 21.7 kDa. MALDI-TOF study showed a peptide sequence of AFCGGSLVNENKMHSAGHCYKSRIQV at the N-Terminal. This sequence recorded 76-84% identity with trypsin from Thunnus thynnus and other fish species. This confirmed that the purified protein was trypsin. The purified enzyme has potential applications in detergent and food industry because of its thermal stability and alkaline nature. PMID:25577096

  14. Community Needs and Barriers to Healthy Dietary Intake and Physical Activity in a Native American Reservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE: Identify unique cultural needs, priorities, program delivery preferences and barriers to achieving a healthy diet and lifestyle in one Native American community. DESIGN: A novel modified nominal group technique (NGT). SETTING: Four community district’s recreation centers. PARTICIPANTS...

  15. Disulfide bridge structure of ascidian trypsin inhibitor I: similarity to Kazal-type inhibitors.

    PubMed

    Kumazaki, T; Ishii, S

    1990-03-01

    The primary structures of ascidian trypsin inhibitors (iso-inhibitors I and II) were reported in the preceding paper (Kumazaki, T. et al. (1990) J. Biochem. 107, 409-413). Both of them have eight half-cystines in a molecule composed of 55 amino acid residues with a sequence showing no extensive homology to other known protease inhibitors. To locate the four disulfide bridges in the molecule, native inhibitor I was digested with thermolysin to yield cystine-containing peptides. The peptides were separated from each other by reversed-phase HPLC. A core peptide still containing six closely located half-cystines (e.g. -Cys-Arg-Cys and -Cys-Cys-) was further digested with Streptomyces griseus trypsin for cleavage of the Arg-Cys bond. On the other hand, the Cys-Cys bond was split by applying manual Edman degradation to the core peptide. Amino acid composition analyses of the resulting cystine peptides allowed us to define the whole disulfide bridge structure in the parent molecule. The topological relation between the disulfide loops and the reactive site suggested that the ascidian trypsin inhibitor may be classified as a member of the Kazal-type inhibitor family. PMID:2111316

  16. 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. PMID:27094449

  17. 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. PMID:26358735

  18. 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)

  19. 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

    2014-01-01

    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. PMID:25501145

  20. Spatial and activity-dependent catecholamine release in rat adrenal medulla under native neuronal stimulation.

    PubMed

    Wolf, Kyle; Zarkua, Georgy; Chan, Shyue-An; Sridhar, Arun; Smith, Corey

    2016-09-01

    Neuroendocrine chromaffin cells of the adrenal medulla in rat receive excitatory synaptic input through anterior and posterior divisions of the sympathetic splanchnic nerve. Upon synaptic stimulation, the adrenal medulla releases the catecholamines, epinephrine, and norepinephrine into the suprarenal vein for circulation throughout the body. Under sympathetic tone, catecholamine release is modest. However, upon activation of the sympathoadrenal stress reflex, and increased splanchnic firing, adrenal catecholamine output increases dramatically. Moreover, specific stressors can preferentially increase release of either epinephrine (i.e., hypoglycemia) or norepinephrine (i.e., cold stress). The mechanism for this stressor-dependent segregated release of catecholamine species is not yet fully understood. We tested the hypothesis that stimulation of either division of the splanchnic selects for epinephrine over norepinephrine release. We introduce an ex vivo rat preparation that maintains native splanchnic innervation of the adrenal gland and we document experimental advantages and limitations of this preparation. We utilize fast scanning cyclic voltammetry to detect release of both epinephrine and norepinephrine from the adrenal medulla, and report that epinephrine and norepinephrine release are regulated spatially and in a frequency-dependent manner. We provide data to show that epinephrine is secreted preferentially from the periphery of the medulla and exhibits a higher threshold and steeper stimulus-secretion function than norepinephrine. Elevated stimulation of the whole nerve specifically enhances epinephrine release from the peripheral medulla. Our data further show that elimination of either division from stimulation greatly attenuated epinephrine release under elevated stimulation, while either division alone can largely support norepinephrine release. PMID:27597763

  1. 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. PMID:24513726

  2. 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

  3. 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. PMID:25901645

  4. Engineering ecotin for identifying proteins with a trypsin fold.

    PubMed

    Sathler, Plínio C; Craik, Charles S; Takeuchi, Toshihiko; Zingali, Russolina B; Castro, Helena C

    2010-04-01

    Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes. PMID:19728173

  5. Express Sequence Tag Analysis - Identification of Anseriformes Trypsin Genes from Full-Length cDNA Library of the Duck (Anas platyrhynchos) and Characterization of Their Structure and Function.

    PubMed

    Yu, Haining; Cai, Shasha; Gao, Jiuxiang; Wang, Chen; Qiao, Xue; Wang, Hui; Feng, Lan; Wang, Yipeng

    2016-02-01

    Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has been used widely in various biotechnological processes. In the current study, a full-length cDNA library with capacity of 5·10(5) CFU/ml from the duck (Anas platyrhynchos) was constructed. Using express sequence tag (EST) sequencing, genes coding two trypsins were identified and two full-length trypsin cDNAs were then obtained by rapid-amplification of cDNA end (RACE)-PCR. Using Blast, they were classified into the trypsin I and II subfamilies, but both encoded a signal peptide, an activation peptide, and a 223-a.a. mature protein located in the C-terminus. The two deduced mature proteins were designated as trypsin-IAP and trypsin-IIAP, and their theoretical isoelectric points (pI) and molecular weights (MW) were 7.99/23466.4 Da and 4.65/24066.0 Da, respectively. Molecular characterizations of genes were further performed by detailed bioinformatics analysis. Phylogenetic analysis revealed that trypsin-IIAP has an evolution pattern distinct from trypsin-IAP, suggesting its evolutionary advantage. Then the duck trypsin-IIAP was expressed in an Escherichia coli system, and its kinetic parameters were measured. The three dimensional structures of trypsin-IAP and trypsin-IIAP were predicted by homology modeling, and the conserved residues required for functionality were identified. Two loops controlling the specificity of the trypsin and the substrate-binding pocket represented in the model are almost identical in primary sequences and backbone tertiary structures of the trypsin families. PMID:27260395

  6. 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. PMID:21861686

  7. Explicit and Implicit Second Language Training Differentially Affect the Achievement of Native-like Brain Activation Patterns

    PubMed Central

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

    2013-01-01

    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. PMID:21861686

  8. 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

  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. 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.

  11. Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.

    PubMed Central

    Schmid, R; Seydl, K; Baumgartner, W; Groschner, K; Romanin, C

    1995-01-01

    The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates. PMID:8580328

  12. 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. PMID:26049798

  13. 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

  14. Trypsin and splanchnic protein turnover during feeding and fasting in human subjects.

    PubMed

    O'keefe, Stephen J D; Lee, Ronzo B; Li, Jing; Zhou, Wen; Stoll, Barbara; Dang, Qianyu

    2006-02-01

    Knowledge of the stimulatory effects of enteral and parenteral (intravenous) feeding on the synthesis and turnover of trypsin would help in the management of acute pancreatitis, because the disease is caused by the premature activation of trypsin. To investigate this, we labeled intravenous infusions with [1-(13)C]leucine and enterals with [(2)H]leucine and measured isotope enrichment of plasma, secreted trypsin, and duodenal mucosal proteins over 6 h by duodenal perfusion/aspiration and endoscopic biopsy. Thirty healthy volunteers were studied during fasting (n = 7), intravenous feeding (n = 6), or postpyloric enteral feeding [duodenal polymeric (n = 6), elemental duodenal (n = 6), and jejunal elemental (n = 5)]. All diets provided 1.5 g x kg(-1) x day(-1) protein and 40 kcal x kg(-1) x day(-1) energy. Results demonstrated that compared with fasting, enteral feeding increased the rate of appearance (71 +/- 4 vs. 91 +/- 5 min, P = 0.01) and secretion (546 +/- 80 vs. 219 +/- 37 U/h, P = 0.01) of newly labeled trypsin and expanded zymogen stores (1,660 +/- 237 vs. 749 +/- 133 units, P = 0.03). These differences persisted whether the feedings were polymeric or elemental, duodenal, or jejunal. In contrast, intravenous feeding had no effect on basal rates. Differential labeling of the plasma amino acid pool by enteral and intravenous isotope infusions suggested that 35% of absorbed amino acids were retained within the splanchnic bed during enteral feeding and that mucosal protein turnover increased from a fasting rate of 34 +/- 6 to 108 +/- 8%/day (P < 0.05) compared with no change after intravenous feeding. In conclusion, all common forms of enteral feeding stimulate the synthesis and secretion of pancreatic trypsin, and only parenteral nutrition avoids it. PMID:16123201

  15. 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.

  16. Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization.

    PubMed

    Malmsten; Larsson

    2000-10-01

    The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads. PMID:10915949

  17. A reinvestigation of a synthetic peptide (TrPepz) designed to mimic trypsin.

    PubMed Central

    Wells, J A; Fairbrother, W J; Otlewski, J; Laskowski, M; Burnier, J

    1994-01-01

    Recently, a 29-residue cyclic peptide was synthesized (TrPepz) that was reported to possess nearly the same catalytic activity and specificity as the pancreatic serine protease, trypsin, for hydrolysis of a small ester substrate, N-tosyl-L-arginine methyl ester (TAME), and small and large peptides [Atassi, M. Z. & Manshouri, T. (1993) Proc. Natl. Acad Sci. USA 90, 8282-8286]. To study these results we have resynthesized TrPepz and a related cyclic peptide reported to possess some trypsin-like activity. The authenticity of each peptide was confirmed by mass spectrometry, peptide sequencing, compositional analysis, and 1H NMR spectroscopy. However, neither peptide exhibited any detectable esterase activity or amidase activity under a variety of conditions tested. Molecular modeling studies indicated it was possible for TrPepz to be nearly superimposed upon the active site of trypsin. However, NMR experiments showed the structure of the cyclic peptide to be disordered. Thus, we were unable to confirm the results of Atassi and Manshouri. Our results are consistent with the view that serine protease activity depends not only on the presence of catalytic groups but also on their precise and stable alignment. PMID:8183880

  18. 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.

  19. Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons.

    PubMed

    Balkowiec, Agnieszka; Katz, David M

    2002-12-01

    Brain-derived neurotrophic factor (BDNF) plays a critical role in activity-dependent modifications of neuronal connectivity and synaptic strength, including establishment of hippocampal long-term potentiation (LTP). To shed light on mechanisms underlying BDNF-dependent synaptic plasticity, the present study was undertaken to characterize release of native BDNF from newborn rat hippocampal neurons in response to physiologically relevant patterns of electrical field stimulation in culture, including tonic stimulation at 5 Hz, bursting stimulation at 25 and 100 Hz, and theta-burst stimulation (TBS). Release was measured using the ELISA in situ technique, developed in our laboratory to quantify secretion of native BDNF without the need to first overexpress the protein to nonphysiological levels. Each stimulation protocol resulted in a significant increase in BDNF release that was tetrodotoxin sensitive and occurred in the absence of glutamate receptor activation. However, 100 Hz tetanus and TBS, stimulus patterns that are most effective in inducing hippocampal LTP, were significantly more effective in releasing native BDNF than lower-frequency stimulation. For all stimulation protocols tested, removal of extracellular calcium, or blockade of N-type calcium channels, prevented BDNF release. Similarly, depletion of intracellular calcium stores with thapsigargin and treatment with dantrolene, an inhibitor of calcium release from caffeine-ryanodine-sensitive stores, markedly inhibited activity-dependent BDNF release. Our results indicate that BDNF release can encode temporal features of hippocampal neuronal activity. The dual requirement for calcium influx through N-type calcium channels and calcium mobilization from intracellular stores strongly implicates a role for calcium-induced calcium release in activity-dependent BDNF secretion. PMID:12451139

  20. 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. PMID:24670137

  1. Legends of Native Americans.

    ERIC Educational Resources Information Center

    Flagg, Ann

    1999-01-01

    Presents a theme unit that includes elementary-level, cross-curricular lessons about lifestyle, belief systems, traditions, and history of Native Americans. The unit includes a poster which offers a traditional Cherokee story, literature on Native American legends, and a variety of cross-curricular activities. The unit ends with students writing…

  2. The presence and inactivation of trypsin inhibitors, tannins, lectins and amylase inhibitors in legume seeds during germination. A review.

    PubMed

    Savelkoul, F H; van der Poel, A F; Tamminga, S

    1992-01-01

    During the germination of legume seeds, enzymes become active in order to degrade starch, storage-protein and proteinaceous antinutritional factors. The degradation of storage-protein is necessary to make peptides and amino acids available in order to stimulate seed growth and early plant growth. Proteinaceous antinutritional factors such as amylase inhibitors, lectins and trypsin inhibitors are present in legume seeds and protect them against predators. However, during germination, they degrade to a lower level by the action of several enzymes. The effect of germination on the content and activity of amylase inhibitors, lectins, tannins and trypsin inhibitors is discussed. PMID:1372122

  3. 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.

  4. 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.

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

    PubMed

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

    2015-09-01

    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

  6. 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

  7. 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.

  8. 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

  9. Affinity chromatography of trypsin and related enzymes. III. Purification of Streptomyces griseus trypsin using an affinity adsorbent containing a tryptic digest of protamine as a ligand.

    PubMed

    Yokosawa, H; Hanba, T; Ishii, S

    1976-04-01

    A new, simple method has been developed for the purification of Streptomyces griseus trypsin [EC 3.4.21.4] from Pronase. Only a single operation of affinity chromatography on an agarose derivative, which was easily prepared by coupling a tryptic digest of salmine to cyanogen bromide-activated Sepharose 4B, was required. A high degree of homogeneity was demonstrated for the purified enzyme by disc electrophoresis, SDS-polyacrylamide gel electrophoresis and gel filtration, as well as by active-site titration. The behavior of a carboxypeptides B [EC 3.4.12.3]-like enzyme present in Pronase is also discussed. PMID:819428

  10. Characterization and immobilization of trypsin on tannic acid modified Fe3O4 nanoparticles.

    PubMed

    Atacan, Keziban; Özacar, Mahmut

    2015-04-01

    Fe3O4 nanoparticles (NPs) were synthesized by co-precipitating Fe2+ and Fe3+ in an ammonia solution. Fe3O4 NPs functionalized with tannic acid were prepared. After functionalization process, trypsin enzyme was immobilized on these Fe3O4 NPs. The influence of pH, temperature, thermal stability, storage time stability and reusability on non-covalent immobilization was studied. The properties of Fe3O4 and its modified forms were examined by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), UV-vis spectrometer (UV) and X-ray diffraction (XRD), magnetization and zeta potential measurements. The immobilized enzyme was slightly more stable than the free enzyme at 45°C. According to the results, the activity of immobilized trypsin was preserved 55% at 45°C after 2 h and 90% after 120 days storage. In addition, the activity of the immobilized trypsin was preserved 40% of its initial activity after eight times of successive reuse. PMID:25686792

  11. Studies on human plasma C1 inactivator-enzyme interactions. I. Mechanisms of interaction with C1s, plasmin, and trypsin.

    PubMed Central

    Harpel, P C; Cooper, N R

    1975-01-01

    This study has explored the nature of the molecular events which occur when C1 inactivator, a human plasma inhibitor of the complement, kinin-forming, coagulation, and fibrinolytic enzyme systems, interacts with C1s, plasmin, and trypsin. Purified inhibitor preparations demonstrated two bands, when examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The molecular weights of the major and minor bands were 105,000 and 96,000 daltons, respectively. The minor component appeared to be immunologically and functionally identical to the main C1 inactivator component. Loss of C1s and plasmin functional activity was associated with the formation of a 1:1 molar complex between the inhibitor and each enzyme. These complexes were stable in the presence of SDS and urea. The light chain of both these enzymes provided the binding site for C1 inactivator. Complex formation and enzyme inhibition occurred only with native and not with an inhibitor preparation denatured by acid treatment, thereby demonstrating the importance of conformational factors in the enzyme-inhibitor reaction. Although peptide bond cleavage of the C1 inactivator molecule by C1s was not documented, plasmin was found to degrade the inhibitor with the production of several characteristic derivatives. At least one of these products retained the ability to complex with C1s and plasmin. Trypsin, which failed to form a complex with C1 inactivator, degraded the inhibitor in a limited and sequential manner with the production of nonfunctional derivatives one of which appeared structurally similar to a plasmin-induced product. These studies therefore, provide new information concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits. Images PMID:123251

  12. Interaction of salivary and midgut proteins of Helicoverpa armigera with soybean trypsin inhibitor.

    PubMed

    Upadhyay, Santosh Kumar; Chandrashekar, Krishnappa

    2012-03-01

    Feeding of Helicoverpa armigera larvae on semi-synthetic diet containing Soybean trypsin inhibitor (STI) resulted in disappearance of STI sensitive protease in salivary and midgut protease extract. This might be due to in situ inhibition by dietary STI. STI was largely degraded within 1 h of incubation with total salivary protease (1:1). Degradation was relatively low in midgut proteases. STI interacting proteins were isolated from saliva and midgut extracts of larvae fed on STI supplemented diet using affinity column. Most of the isolated proteins showed caseinolytic activity in zymogram. Denovo sequencing data of seven different peptides selected from trypsin digested total protein showed similarity to chymotrypsinogen, serine protease, aminopeptidase N, peroxidase, hypothetical protein and muscle specific protein. PMID:22415700

  13. Ethylene glycol and the thermostability of trypsin in a reverse micelle system.

    PubMed

    Stupishina, E A; Khamidullin, R N; Vylegzhanina, N N; Faizullin, D A; Zuev, Yu F

    2006-05-01

    The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms. PMID:16732732

  14. 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

  15. 75 FR 68026 - Native American CDFI Assistance (NACA) Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-04

    ... Community Development Financial Institutions Fund Native American CDFI Assistance (NACA) Program Funding... the Native American CDFI Assistance (NACA) Program. Announcement Type: Announcement of funding... direct at least 50 percent of their activities toward serving Native American, Alaska Native,...

  16. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

    PubMed Central

    Valdes-Rodriguez, S; Segura-Nieto, M; Chagolla-Lopez, A; Verver y Vargas-Cortina, A; Martinez-Gallardo, N; Blanco-Labra, A

    1993-01-01

    A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima. PMID:8290633

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

    PubMed Central

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

    2007-01-01

    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. PMID:17610323

  18. 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

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

    PubMed

    Utama, Dicky Tri; Lee, Seung Gyu; Baek, Ki Ho; Kim, Hye-Kyung; Cho, Chang-Yeon; Lee, Cheol-Koo; Lee, Sung Ki

    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

  20. 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. PMID:26169062

  1. Trypsin inhibitor from 3 legume seeds: fractionation and proteolytic inhibition study.

    PubMed

    Wati, Richa Kusuma; Theppakorn, Theerapong; Benjakul, Soottawat; Rawdkuen, Saroat

    2010-04-01

    The trypsin inhibitor from navy beans (Phaseoulus vulgaris), red kidney beans (Phaseoulus vulgaris L.), and adzuki beans (Vigna angularis) provided by the Royal Project Foundation in Thailand was isolated by heat and ammonium sulfate (AS) precipitation. Incubation at 70 degrees C for 10 min produced the highest trypsin inhibitor recovery for all legumes. The AS precipitation with 60% to 80% saturation (precipitate IV) resulted in 41-, 88-, and 34-fold of the purity and (-)26%, 126%, and (-)47% of percentage of activity increase for navy beans, red kidney beans, and adzuki beans, respectively. The trypsin inhibitors had a molecular weight of 132 kDa for navy beans, 118 kDa for red kidney beans, and 13 kDa for adzuki beans under nonreducing conditions. The obtained precipitate IV fraction from each legume effectively prevented the degradation of the tilapia muscle with concentration dependent. The myosin heavy chain increased as the concentration of the inhibitor fraction increased, especially at the highest level of addition. The result indicated that the precipitate IV from these legumes have potential for use as a protease inhibitor in fishery related products. PMID:20492270

  2. The release of rat intestinal cholecystokinin after oral trypsin inhibitor measured by bio-assay.

    PubMed Central

    Brand, S J; Morgan, R G

    1981-01-01

    The distribution, molecular form and release of cholecystokinin (CCK)-like activity in extracts of rat small intestine was studied with an in vitro gall-bladder bio-assay. In contrast to the reported heterogeneity of CCK-like immunoreactivity in the intestine, only a single molecular form of CCK-like activity was detected using the bio-assay. 2. The CCK-like activity eluted from Sephadex G50 with a Kav of 0.69, after the triacontriapeptide of cholecystokinin (CCK33) and before cholecystokinin octapeptide 2500, may represent the 22 amino acid peptide of CCK (CCK22). The bio-assay peak of CCK-like activity had pancreozymin activity and CCK/gastrin C terminal immunoreactivity. The CCK-like activity weas readily extracted from the small intestine at neutral pH, but subsequent treatment with cold 0.5 M-acetic acid extracted further CCK-like activity of the same molecular form as that recovered under neutral conditions. 3. The bio-assay detected no CCK-like activity, nor was pancreozymin-like activity found in fractions corresponding to CCK33 or CCK8 after Sephadex G50 chromatography of rat intestinal extracts. 4. Oral trypsin inhibitor was a potent stimulus for the release of CCK-like activity from the upper small intestine of the rat. After oral trypsin inhibitor release, CCK-like activity was rapidly resynthesized. PMID:7320918

  3. 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

  4. Inhibitory activity of thymol on native and mature Gardnerella vaginalis biofilms: in vitro study.

    PubMed

    Braga, Pier Carlo; Dal Sasso, Monica; Culici, Maria; Spallino, Alessandra

    2010-01-01

    Bacterial vaginosis (BV) is the most frequent diagnosis made in women with lower genital tract symptoms. It has recently been observed that 90 % of subjects with BV show the growth of bacteria in the form of biofilms as against only 10% without BV, and that Gardnerella vaginalis was the predominant species. The propensity of G. vaginalis to form biofilm is clinically relevant because this form of growth allows it to tolerate higher concentrations of certain antibiotics, thus increasing the possibilty of recurrent BV even after apparently curative therapy. The aim of this study was to investigate whether thymol (CAS 89-83-8), a molecule present in thyme essential oil, that is credited with having a series of pharmacological properties including antimicrobial and antifungal effects, can interfere with newly formed and mature G. vaginalis biofilms. The ability of G. vaginalis ATCC 49145 and two G. vaginalis strains isolated from human BV to form biofilm in flat-bottomed 96-well microtitre plates was verified, and the effects of thymol concentrations ranging from 1 to 1/16 MIC (minimum inhibitory concentration) on preformed and mature biofilms was investigated by means of spectrophotometric analysis, Nomarski interference contrast microscopy, and fluorescence microscopy with live-dead cell visualisation (SYTO 9 and propidium iodide). Native biofilm was inhibited by concentrations ranging from 1 MIC to 1/8 MIC (32.77% +/- 2.37 to 11.39% +/- 1.46), and mature biofilm was inhibited by concentrations ranging from 1 MIC to 1/4 MIC (26.18% +/- 1.36 to 13.20% +/- 1.44). Nomarski interference contrast and fluorescence microscopy visually confirmed these findings. As biofilm is a multi-factorial phenomenon, the multiple mechanisms of thymol may act on different steps in the evolution of mature biofilm. PMID:21175040

  5. 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

  6. Characterization of a trypsin inhibitor from equine urine.

    PubMed

    Veeraragavan, K; Singh, K; Wachter, E; Hochstrasser, K

    1992-03-01

    A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine. PMID:1627153

  7. 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

    2016-01-01

    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. PMID:26805792

  8. Weight-bearing activity and foot parameters in Native Americans with diabetes with and without foot sensation.

    PubMed

    Cuaderes, Elena; Lamb, Lyndon; Khan, Myrna; Lawrence, Gary

    2009-01-01

    Amputations from diabetic foot ulcers account for substantial health care costs and pain. At the point of neuropathy, it has been recommended that weight-bearing activities (WBA) be restricted to reduce the risk of ulcers. While regular WBA has been shown to control glycemia, stress from this exercise may lead to ulcers by various processes. This investigation compared Native Americans with diabetes, with or without foot sensation and who reported regular or no leisure-time WBA, with variables of significance for risk of diabetic foot ulcers. Analysis revealed that there were no differences in barefoot standing plantar pressure or plantar skin hardness between the four groups. Further study is recommended with a larger sample and the use of instruments that are more valid and reliable. PMID:20669398

  9. 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. PMID:23229153

  10. 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

  11. Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, aminoterminal sequence determination, and cloning and sequencing of the gene.

    PubMed

    Noriega, F G; Wang, X Y; Pennington, J E; Barillas-Mury, C V; Wells, M A

    1996-02-01

    Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. It plays an essential role in the transcriptional activation of the late trypsin form, the major midgut endoprotease involved in the blood meal digestion. Early trypsin is the most abundant midgut polypeptide isolated by benzamidine-sepharose affinity chromatography 3 h after feeding. The amino-terminal sequence of the early trypsin protein matches that of the 3a1 cDNA for a putative trypsinogen described by Kalhok et al. (Insect. Molec. Biol., 2, 71-79, 1993). The early trypsin cDNA was over expressed in Escherichia coli. Polyclonal antibodies generated against this recombinant protein were used to show that the enzyme was present in the midgut during the first 4 h after feeding. A 2.5 kb genomic clone of the early trypsin was isolated, mapped and subcloned. A 1.56 kb subclone, corresponding to 1303 bp of the upstream regulatory region and 265 bp of the coding region, was sequenced. The gene contains a 64 nucleotide intron which interrupts the codon for Val at position 18 of the protein. This Val is located toward the end of the putative signal sequence of the protein. PMID:8882654

  12. New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket.

    PubMed

    Perera, Erick; Pons, Tirso; Hernandez, Damir; Moyano, Francisco J; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2010-09-01

    Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed. PMID:20649906

  13. Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity.

    PubMed

    Nauze, Michel; Gonin, Lauriane; Chaminade, Brigitte; Perès, Christine; Hullin-Matsuda, Francoise; Perret, Bertrand; Chap, Hugues; Gassama-Diagne, Ama

    2002-11-15

    Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH(2)-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH(2)-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein. PMID:12194976

  14. Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth.

    PubMed

    Espinoza, I; Galindo, M; Bizarro, C V; Ferreira, H B; Zaha, A; Galanti, N

    2005-11-01

    Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms. PMID:15887242

  15. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  16. Characterization of cellulolytic activities of environmental bacterial consortia from an Argentinian native forest.

    PubMed

    Romano, Nelson; Gioffré, Andrea; Sede, Silvana M; Campos, Eleonora; Cataldi, Angel; Talia, Paola

    2013-08-01

    Cellulolytic activities of three bacterial consortia derived from a forest soil sample from Chaco region, Argentina, were characterized. The phylogenetic analysis of consortia revealed two main highly supported groups including Achromobacter and Pseudomonas genera. All three consortia presented cellulolytic activity. The carboxymethylcellulase (CMCase) and total cellulase activities were studied both quantitatively and qualitatively and optimal enzymatic conditions were characterized and compared among the three consortia. Thermal and pH stability were analyzed. Based on its cellulolytic activity, one consortium was selected for further characterization by zymography. We detected a specific protein of 55 kDa with CMCase activity. In this study, we have shown that these consortia encode for cellulolytic enzymes. These enzymes could be useful for lignocellulosic biomass degradation into simple components and for different industrial applications. PMID:23471693

  17. 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

  18. Myofibroblastic cell activation and neovascularization predict native liver survival and development of esophageal varices in biliary atresia

    PubMed Central

    Suominen, Janne S; Lampela, Hanna; Heikkilä, Päivi; Lohi, Jouko; Jalanko, Hannu; Pakarinen, Mikko P

    2014-01-01

    myofibroblastic cell activation, fibrogenesis and neovascularization are enhanced in BA, progress with increasing PE age and relate to native liver survival and development of esophageal varices. PMID:24696612

  19. Phytochemistry and biologic activities of caulerpa peltata native to oman sea.

    PubMed

    Movahhedin, Nasrin; Barar, Jaleh; Fathi Azad, Fatemeh; Barzegari, Abolfazl; Nazemiyeh, Hossein

    2014-01-01

    General toxicity, antiproliferative, antibacterial and antioxidant activities of Caulerpa peltata J.V.Lamouroux (Caulerpaceae) collected from Oman Sea were investigated. Dried, ground alga was Soxhlet-extracted with hexane, dichloromethane and methanol successively. The methanol extract was subjected to vacuum liquid chromatography (VLC) fractionation on silica gel using a step gradient of different mixture of solvents. A known alkaloid, caulerpin, was subsequently isolated from the fraction eluted by ethyl acatete100%. The antioxidant activity of all extracts was assessed by using the (DPPH) assay. Antiproliferative activity of the all extracts and caulerpin against the cancerous cell line was evaluated using MTT assay. General toxicity of extracts was determined using Brine Shrimp Lethality Assay (BSLA). Based on our results, a weak activity observed for all extracts in MTT assay, while they were toxic toward brine shrimp nauplii comparing to the podophylotoxin. This is the first report on phytochemistry and bioactivity of C. peltata which collected from Oman Sea. PMID:25237346

  20. Phytochemistry and Biologic Activities of Caulerpa Peltata Native to Oman Sea

    PubMed Central

    Movahhedin, Nasrin; Barar, Jaleh; Fathi Azad, Fatemeh; Barzegari, Abolfazl; Nazemiyeh, Hossein

    2014-01-01

    General toxicity, antiproliferative, antibacterial and antioxidant activities of Caulerpa peltata J.V.Lamouroux (Caulerpaceae) collected from Oman Sea were investigated. Dried, ground alga was Soxhlet-extracted with hexane, dichloromethane and methanol successively. The methanol extract was subjected to vacuum liquid chromatography (VLC) fractionation on silica gel using a step gradient of different mixture of solvents. A known alkaloid, caulerpin, was subsequently isolated from the fraction eluted by ethyl acatete100%. The antioxidant activity of all extracts was assessed by using the (DPPH) assay. Antiproliferative activity of the all extracts and caulerpin against the cancerous cell line was evaluated using MTT assay. General toxicity of extracts was determined using Brine Shrimp Lethality Assay (BSLA). Based on our results, a weak activity observed for all extracts in MTT assay, while they were toxic toward brine shrimp nauplii comparing to the podophylotoxin. This is the first report on phytochemistry and bioactivity of C. peltata which collected from Oman Sea. PMID:25237346

  1. 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. PMID:26123168

  2. 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. PMID:26615146

  3. Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

    PubMed

    Austin, S; Buck, M; Cannon, W; Eydmann, T; Dixon, R

    1994-06-01

    The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA. PMID:8206822

  4. 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. PMID:25857980

  5. RENAL INSUFFICIENCY FOLLOWING TRYPSIN INJECTION INTO THE RENAL ARTERIES.

    PubMed

    Friedman, M; Katz, L N

    1938-09-30

    1. The injection of trypsin into both renal arteries of the dog was found to cause an acute necrosis of large sections of the kidney, an immediate excretory insufficiency, and a transient hypertension. 2. Dogs surviving the acute phase of the trypsin injection, developed a chronic renal excretory insufficiency with no hypertension, despite the severity and duration of the renal excretory insufficiency. 3. The application of a Goldblatt clamp to the renal artery of one of the two kidneys, previously injected with trypsin, led to a rise in blood pressure which returned at once to normal when the ischemic kidney was removed, even though the pre-existing renal excretory insufficiency was augmented. This experience demonstrated unequivocally that chronic renal excretory insufficiency and hypertension are not directly related. 4. The application of a Goldblatt clamp to the renal artery of one kidney and the simultaneous injection of trypsin into the other led to a hypertension. The later removal of the ischemic kidney led to a severe renal excretory insufficiency, at the same time the pre-existing hypertension disappeared. This indicated again that renal excretory insufficiency and renal ischemia produced different phenomena and that the former had no direct relation to hypertension. PMID:19870800

  6. Some physical and chemical properties of trypsin-digested nucleoprotein

    PubMed Central

    Marks, Dawn B.; Schumaker, Verne N.

    1968-01-01

    A DNA–peptide complex that is soluble in 0·2m-sodium chloride can be prepared by trypsin digestion of calf thymus nucleoprotein. The trypsin-digested nucleoprotein molecule contains about 70% of DNA and 30% of peptides by weight, and consists of one DNA molecule associated with arginine-rich peptides. A series of trypsin-digested nucleoprotein preparations differing only in molecular weight were prepared by blending. The intrinsic viscosity and average sedimentation coefficient were determined for each of these preparations. Then the DNA was isolated from each preparation and the hydrodynamic measurements were repeated on the DNA. From a comparison of these results it was concluded that the presence of the complex-forming peptides causes a large decrease in intrinsic viscosity of the DNA and an increase in sedimentation coefficient. In addition, the hydrodynamic data indicate that the DNA–peptide complex behaves like a coil in solution but is more compact than the same length of DNA. The `melting' profiles, streptomycin precipitation curves and maximum viscosities obtained with ethidium bromide binding for the trypsin-digested nucleoprotein are similar to those of purified DNA, and markedly different from those of undigested nucleoprotein. These findings suggest that the peptides are not strongly associated with the DNA, and that secondary valency forces are involved in the binding. PMID:5683510

  7. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Trypsin test system. 862.1725 Section 862.1725 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis...

  8. 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... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis...

  9. 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... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis...

  10. 21 CFR 862.1725 - Trypsin test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Trypsin test system. 862.1725 Section 862.1725 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis...

  11. 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.

  12. Art Activities to Improve Self-Esteem among Native Hawaiian Children.

    ERIC Educational Resources Information Center

    Omizo, Michael M.; Omizo, Sharon A.

    1989-01-01

    Investigated effects of group counseling using art activities in improving self-esteem among Hawaiian elementary children (N=50). Found subjects who participated in counseling had higher Social Peer-Related and Academics/School-Related Self-Esteem scores than children who did not participate. (ABL)

  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. 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. PMID:25701144

  15. Low affinity block of native and cloned hyperpolarization-activated Ih channels by Ba2+ ions.

    PubMed

    van Welie, Ingrid; Wadman, Wytse J; van Hooft, Johannes A

    2005-01-10

    Ba2+ is commonly used to discriminate two classes of ion currents. The classical inward-rectifying K+ current, I(Kir), is blocked by low millimolar concentrations of Ba2+, whereas the hyperpolarization-activated cation current, I(h), is assumed not to be sensitive to Ba2+. Here we investigated the effects of Ba2+ on I(h) currents recorded from rat hippocampal CA1 pyramidal neurons, and on cloned I(h) channels composed of either HCN1 or HCN2 subunits transiently expressed in Human Embryonic Kidney (HEK) 293 cells. The results show that low millimolar concentrations of Ba2+ reduce the maximal I(h) conductance (IC50 approximately 3-5 mM) in both CA1 pyramidal neurons and in HEK 293 cells without specificity for HCN1 or HCN2 subunits. In addition, Ba2+ decreases the rate of activation and increases the rate of deactivation of I(h) currents. Neither the half-maximal voltage of activation, V(h), nor the reversal potential of the I(h) channels were affected by Ba2+. The combined results suggest that B2+, at concentrations commonly used to block I(Kir) currents, also reduces the conductance of I(h) channels without subunit specificity, and affects the kinetics of I(h) channel gating. PMID:15659289

  16. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Champion, C I; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1996-01-01

    The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete. PMID:8759855

  17. Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens

    PubMed Central

    Mason, Kevin M.; Dryden, Tricia D.; Bigley, Nancy J.; Fink, Pamela S.

    1998-01-01

    Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+ T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo. PMID:9784507

  18. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Inter-alpha trypsin inhibitor immunological test... Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An inter-alpha trypsin inhibitor immunological test system is a device that consists of the reagents used...

  19. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Liquid crystalline trypsin, Peru balsam, castor... NEW ANIMAL DRUGS § 524.2620 Liquid crystalline trypsin, Peru balsam, castor oil. (a) Specifications—(1) Each gram of liquid or aerosol contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of...

  20. Structural determinants of trypsin affinity and specificity for cationic inhibitors.

    PubMed Central

    Polticelli, F.; Ascenzi, P.; Bolognesi, M.; Honig, B.

    1999-01-01

    The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed. PMID:10631977

  1. Structural determinants of trypsin affinity and specificity for cationic inhibitors.

    PubMed

    Polticelli, F; Ascenzi, P; Bolognesi, M; Honig, B

    1999-12-01

    The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed. PMID:10631977

  2. 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...

  3. 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

  4. Circular proteins in plants: solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis.

    PubMed

    Felizmenio-Quimio, M E; Daly, N L; Craik, D J

    2001-06-22

    Much interest has been generated by recent reports on the discovery of circular (i.e. head-to-tail cyclized) proteins in plants. Here we report the three-dimensional structure of one of the newest such circular proteins, MCoTI-II, a novel trypsin inhibitor from Momordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small beta-sheet, several turns, and a cystine knot arrangement of the three disulfide bonds. Interestingly, the molecular topology is similar to that of the plant cyclotides (Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) J. Mol. Biol. 294, 1327-1336), which derive from the Rubiaceae and Violaceae plant families, have antimicrobial activities, and exemplify the cyclic cystine knot structural motif as part of their circular backbone. The sequence, biological activity, and plant family of MCoTI-II are all different from known cyclotides. However, given the structural similarity, cyclic backbone, and plant origin of MCoTI-II, we propose that MCoTI-II can be classified as a new member of the cyclotide class of proteins. The expansion of the cyclotides to include trypsin inhibitory activity and a new plant family highlights the importance and functional variability of circular proteins and the fact that they are more common than has previously been believed. Insights into the possible roles of backbone cyclization have been gained by a comparison of the structure of MCoTI-II with the homologous acyclic trypsin inhibitors CMTI-I and EETI-II from the Cucurbitaceae plant family. PMID:11292835

  5. 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.

  6. Chemical composition and antioxidant activity of essential oil of six pinus taxa native to China.

    PubMed

    Xie, Qing; Liu, Zhihong; Li, Zhouqi

    2015-01-01

    The essential oils obtained by steam distillation from needles of six China endemic Pinus taxa (P. tabulaeformis, P. tabulaeformis f. shekanensis, P. tabulaeformis var. mukdensis, P. tabulaeformis var. umbraculifera, P. henryi and P. massoniana) were analysed by GC/MS. A total of 72 components were separated and identified by GC/MS from the six taxa. The major constituents of the essential oils were: α-pinene (6.78%-20.55%), bornyl acetale (3.32%-12.71%), β-caryophellene (18.26%-26.31%), α-guaiene (1.23%-8.19%), and germacrene D (1.26%-9.93%). Moreover, the essential oils were evaluated for antioxidant potential by three assays (DPPH, FRAP and ABTS) and tested for their total phenolic content. The results showed that all essential oils exhibited acceptable antioxidant activities and these strongly suggest that these pine needles may serve as a potential source of natural antioxidants for food and medical purposes. PMID:26007189

  7. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

    PubMed Central

    Radisky, Evette S.; Lee, Justin M.; Lu, Chia-Jung Karen; Koshland, Daniel E.

    2006-01-01

    Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction. PMID:16636277

  8. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

    PubMed Central

    Milgrom, Y M; Murataliev, M B; Boyer, P D

    1998-01-01

    Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid. PMID:9480927

  9. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

    PubMed

    Matveev, Vladimir V

    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

  10. 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

  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 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…

  13. SdPI, The First Functionally Characterized Kunitz-Type Trypsin Inhibitor from Scorpion Venom

    PubMed Central

    Li, Tian; He, Yawen; Ma, Yibao; Chen, Zongyun; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

    2011-01-01

    Background Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized. Principal Findings A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (Ki = 1.6×10−7 M) and thermostability. Conclusions The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides. PMID:22087336

  14. 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

  15. 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. PMID:19431098

  16. Systemic lupus erythematosus: molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA2-Me-pro-ChTr possessing two different activities-trypsin-like and metalloprotease.

    PubMed

    Timofeeva, Anna M; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

    2015-12-01

    Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies. PMID:26174315

  17. In vitro activity on human gut bacteria of murta leaf extracts (Ugni molinae turcz.), a native plant from Southern Chile.

    PubMed

    Shene, Carolina; Canquil, Nelly; Jorquera, Milko; Pinelo, Manuel; Rubilar, Mónica; Acevedo, Francisca; Vergara, Carola; von Baer, Dietrich; Mardones, Claudia

    2012-06-01

    Despite the fact that murta infusions have been used to treat gut/urinary infections by native Chileans for centuries, the mechanisms promoting such effects still remain unclear. As a first attempt to unravel these mechanisms, human fecal samples were incubated in a medium containing water extract of murta leaves (ML) and the growth of different bacterial groups was evaluated. Control incubations were made in media containing fructooligosaccharides (FOS) and glucose as a carbon source. Phenolic compounds in the ML extract, likely promoters of bioactivity, were identified by HPLC-DAD-MS(n) . Concentrations (log₁₀ CFU/mL) of bifidobacteria and lactobacilli in media containing the extract and FOS were 7.33 ± 0.05/4.95 ± 0.20 and 6.44 ± 0.22/6.05 ± 0.06, respectively. Clostridia, anaerobes and Enterobacteriaceae grew to a similar extent in media containing murta extract and FOS. In vitro tests (disk diffusion) showed that Gram-positive (Bacillus and Paenibacillaceae) and Gram-negative (Enterobacteriaceae) bacteria isolated from fecal samples were sensitive to both water and 50/50 ethanol/water extracts of ML (28.4 μg gallic acid equivalents). At this concentration, the antimicrobial activity of ML extracts was significantly (P < 0.05) lower than that of penicillin (10 U), whereas the difference between activity of ML extracts and gentamicine (10 μg) was no significant (P > 0.05). No evidence of dependency between the antimicrobial activity of ML extracts and the enzymatic capability of the sensitive strains was found. PMID:22583138

  18. Estimating cotinine associations and a saliva cotinine level to identify active cigarette smoking in alaska native pregnant women.

    PubMed

    Smith, Julia J; Robinson, Renee F; Khan, Burhan A; Sosnoff, Connie S; Dillard, Denise A

    2014-01-01

    Studies indicate nicotine metabolism varies by race and can change during pregnancy. Given high rates of tobacco use and limited studies among Alaska Native (AN) women, we estimated associations of saliva cotinine levels with cigarette use and second-hand smoke (SHS) exposure and estimated a saliva cotinine cutoff to distinguish smoking from non-smoking pregnant AN women. Using questionnaire data and saliva cotinine, we utilized multi-variable linear regression (n = 370) to estimate cotinine associations with tobacco use, SHS exposure, demographic, and pregnancy-related factors. Additionally, we estimated an optimal saliva cotinine cutoff for indication of active cigarette use in AN pregnant women using receiver operating characteristic (ROC) curve analysis (n = 377). Saliva cotinine significantly decreased with maternal age and significantly increased with cigarettes smoked per day, SHS exposure, and number of previous full term pregnancies. Using self-reported cigarette use in the past 7 days as indication of active smoking, the area under the ROC curve was 0.975 (95 % CI: 0.960-0.990). The point closest to 100 % specificity and sensitivity occurred with a cotinine concentration of 1.07 ng/mL, which corresponded to sensitivity of 94 % and specificity of 94 %. We recommend using a saliva cotinine cutoff of 1 ng/mL to distinguish active smoking in pregnant AN women. This cutoff is lower than used in other studies with pregnant women, most likely due to high prevalence of light or intermittent smoking in the AN population. Continued study of cotinine levels in diverse populations is needed. PMID:23423858

  19. Bio-potency of a 21 kDa Kunitz-type trypsin inhibitor from Tamarindus indica seeds on the developmental physiology of H. armigera.

    PubMed

    Pandey, Prabhash K; Jamal, Farrukh

    2014-11-01

    A trypsin inhibitor purified from the seeds of Tamarindus indica by Sephadex G-75, DEAE-Sepharose and Trypsin-Sepharose CL-4B columns was studied for its antifeedant, larvicidal, pupicidal and growth inhibitory activities against Helicoverpa armigera larvae. Tamarindus trypsin inhibitor (TTI) exhibited inhibitory activity towards total gut proteolytic enzymes of H. armigera (~87%) and bovine trypsin (~84%). Lethal doses which caused mortality and weight reduction by 50% were 1% w/w and 0.50% w/w, respectively. IC50 of TTI against Helicoverpa midgut proteases and bovine trypsin were ~2.10 µg/ml and 1.68 µg/ml respectively. In larval feeding studies the 21 kDa Kunitz-type protein was found to retard growth and development, prolonged the larval-pupal development durations along with adversely affecting the fertility and fecundity of H. armigera. In artificial diet at 0.5% w/w TTI, the efficiency of conversion of ingested food as well as of digested food, relative growth rate, growth index declined whereas approximate digestibility, metabolic cost, relative consumption rate, consumption index and total developmental period enhanced for H. armigera larvae. These results suggest that TTI has toxic and adverse effect on the developmental physiology of H. armigera and could be useful in controlling the pest H. armigera. PMID:25454525

  20. 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

  1. The chemical properties and functional role of a lysine residue within the active site of native sodium and potassium ion-activated adenosinetriphosphatase

    SciTech Connect

    Xu, K.Y.

    1988-01-01

    The peptide, HLLVMKGAPER, which contains Lysine 501 of the {alpha} polypeptide can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion. An immunoadsorbent directed against the carboxy-terminal, -GAPER, has been constructed. Sealed, right-side-out vesicles, prepared from canine renal kidneys, were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin, respectively. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digest obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction it could be concluded that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.

  2. 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…

  3. 21 CFR 184.1914 - Trypsin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the...

  4. Native Skies

    NASA Astrophysics Data System (ADS)

    Benningfield, Damond

    2001-03-01

    People native to North America practiced their own version of astronomy. They tracked the motions of the Sun to help them decide when to plant crops, move their camps, and stage sacred rituals. Some tribes built great circles of stones to help them predict the changing seasons. Others built great mounds of earth to reflect the patterns they saw in the heavens and to align their ceremonial centers with the Sun and the Moon.

  5. Kinetic assessment and effect on developmental physiology of a trypsin inhibitor from Eugenia jambolana (Jambul) seeds on Helicoverpa armigera (Hübner).

    PubMed

    Singh, Dushyant; Jamal, Farrukh; Pandey, Prabhash K

    2014-02-01

    A trypsin inhibitor was purified from the seeds of Eugenia jambolana (Jambul) with a fold purification of 14.28 and a yield recovery of 2.8%. Electrophoretic analysis of E. jambolana trypsin inhibitor (EjTI) revealed a molecular weight of approximately 17.4 kDa on 12% denaturing polyacrylamide gel electrophoresis with or without reduction. EjTI exhibited high stability over a wide range of temperatures (4-80 °C for 30 min) and pH (3.0-10.0) and inhibited trypsin-like activities of the midgut proteinases of fourth instar Helicoverpa armigera larvae by approximately 86%. Feeding assays containing 0.05, 0.15, and 0.45 (% w/w) EjTI on functionally important fourth-instar larvae indicated a dose-dependent downfall in the larval body weight as well as on extent of survival. The nutritional analysis suggests that EjTI exerts toxic effects on H. armigera. Dixon plot analysis revealed competitive inhibition of larval midgut proteinases by EjTI, with an inhibition constant (Ki ) of approximately 3.1 × 10(-9) M. However, inhibitor kinetics using double reciprocal plots for trypsin inhibition demonstrated a mixed inhibition pattern. These observations suggest the potential of E. jambolana trypsin inhibitor protein in insect pest management. PMID:24436204

  6. Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

    PubMed Central

    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. PMID:26121040

  7. NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells

    PubMed Central

    Park, Il Hwan; Hwang, Hye Mi; Jeon, Byeong Hwa; Kwon, Hyung-Joo; Hoe, Kwang Lae; Kim, Young Myeong; Ryoo, Sungwoo

    2015-01-01

    Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated h

  8. 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…

  9. Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.

    PubMed

    Meissner, G; Rousseau, E; Lai, F A

    1989-01-25

    The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel. PMID:2536370

  10. 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. PMID:25678063

  11. Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

    PubMed

    Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

    2016-04-01

    The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. PMID:26947160

  12. A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping.

    PubMed

    Kori, Yekaterina; Patel, Rekha; Neill, Alyssa; Liu, Hongcheng

    2016-01-15

    Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC-MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures. PMID:26720699

  13. The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

    PubMed Central

    Johnson, Paley; Whateley, Tony L.

    1981-01-01

    The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol−1·dm3·s−1 for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol−1·dm3·s−1 for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules

  14. 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

  15. Silica nanoparticles for separation of biologically active amines by capillary electrophoresis with laser-induced native fluorescence detection.

    PubMed

    Kuo, I-Ting; Huang, Yu-Fen; Chang, Huan-Tsung

    2005-06-01

    This paper describes the analysis of biologically active amines by capillary electrophoresis (CE) in conjunction with laser-induced native fluorescence detection. In order to simultaneously analyze amines and acids as well as to achieve high sensitivity, 10 mM formic acid solutions (pH < 4.0) containing silica nanoparticles (SiNPs) were chosen as the background electrolytes. With increasing SiNP concentration, the migration times for seven analytes decrease as a result of increase in electroosmotic flow (EOF) and decrease in their electrophoretic mobilities against EOF. A small EOF generated at pH 3.0 reveals adsorption of SiNPs on the deactivated capillary wall. The decreases in electrophoretic mobilities with increasing SiNP concentration up to 0.3x indicate the interactions between the analytes and the SiNPs. Having a great sensitivity (the limits of detection at a signal-to-noise ratio (S/N) = 3 of 0.09 nM for tryptamine (TA)), high efficiency, and excellent reproducibility (less than 2.4% of the migration times), this developed method has been applied to the analysis of urinal samples with the concentrations of 0.50 +/- 0.02 microM, 0.49 +/- 0.04 microM, and 74 +/- 2 microM for TA, 5-hydroxytryptamine, and tryptophan, respectively. The successful examples demonstrated in this study open up a possibility of using functional nanoparticles for the separation of different analytes by CE. PMID:15937981

  16. Tyrosine sulfation of human trypsin steers S2' subsite selectivity towards basic amino acids.

    PubMed

    Szabó, András; Salameh, Moh'd A; Ludwig, Maren; Radisky, Evette S; Sahin-Tóth, Miklós

    2014-01-01

    Human cationic and anionic trypsins are sulfated on Tyr154, a residue which helps to shape the prime side substrate-binding subsites. Here, we used phage display technology to assess the significance of tyrosine sulfation for the specificity of human trypsins. The prime side residues P1'-P4' in the binding loop of bovine pancreatic trypsin inhibitor (BPTI) were fully randomized and tight binding inhibitor phages were selected against non-sulfated and sulfated human cationic trypsin. The selection pattern for the two targets differed mostly at the P2' position, where variants selected against non-sulfated trypsin contained primarily aliphatic residues (Leu, Ile, Met), while variants selected against sulfated trypsin were enriched also for Arg. BPTI variants carrying Arg, Lys, Ile, Leu or Ala at the P2' position of the binding loop were purified and equilibrium dissociation constants were determined against non-sulfated and sulfated cationic and anionic human trypsins. BPTI variants harboring apolar residues at P2' exhibited 3-12-fold lower affinity to sulfated trypsin relative to the non-sulfated enzyme, whereas BPTI variants containing basic residues at P2' had comparable affinity to both trypsin forms. Taken together, the observations demonstrate that the tyrosyl sulfate in human trypsins interacts with the P2' position of the substrate-like inhibitor and this modification increases P2' selectivity towards basic side chains. PMID:25010489

  17. Expression and purification of the trypsin inhibitor from tartary buckwheat in Pichia pastoris and its novel toxic effect on Mamestra brassicae larvae.

    PubMed

    Ruan, Jingjun; Yan, Jun; Hou, Shengqi; Chen, Hui; Wu, Qi; Han, Xueyi

    2015-01-01

    The gene of the trypsin inhibitor of tartary buckwheat (Fagopyrum tataricum) was successfully cloned, expressed in Pichia pastoris and tested for regulatory effects on insect growth. The three significant factors were optimized by single-factor experiments and central composite design in response surface methodology. Proteins were efficiently expressed at levels of 489.6-527.4 U/mg in shaken flasks. The trypsin inhibitor from tartary buckwheat (FtTI) was purified by affinity chromatography and centrifugal ultrafiltration. The purified FtTI efficiently inhibited trypsin protease activity by competitive inhibition with a Ki value 1.5 nM. The molecular mass of the purified protein was approximately 13.8 kDa. FtTI had a higher toxic killing effect on Mamestra brassicae larvae. The median lethal concentration for the larvae was 15 μg/mL. PMID:25258121

  18. 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. PMID:857902

  19. Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment.

    PubMed

    Slootweg, Jack C; Peters, Nienke; Quarles van Ufford, H Linda C; Breukink, Eefjan; Liskamp, Rob M J; Rijkers, Dirk T S

    2014-10-01

    The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition 'click' chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the 'staples' as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere. PMID:25199583

  20. 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.

  1. 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.

  2. Invasive non-native plants have a greater effect on neighbouring natives than other non-natives.

    PubMed

    Kuebbing, Sara E; Nuñez, Martin A

    2016-01-01

    Human activity is creating a global footprint by changing the climate, altering habitats and reshuffling the distribution of species. The movement of species around the globe has led to the naturalization and accumulation of multiple non-native species within ecosystems, which is frequently associated with habitat disturbance and changing environmental conditions. However, interactions among species will also influence community composition, but little is known about the full range of direct and indirect interactions among native and non-native species. Here, we show through a meta-analysis of 1,215 pairwise plant interactions between 274 vascular plant species in 21 major habitat types that interactions between non-native plants are asymmetrical with interactions between non-native and native plants. Non-native plants were always bad neighbours, but the negative effect of non-natives on natives was around two times greater than the effect of non-natives on other non-natives. In contrast, the performance of non-native plants was five times higher in the presence of a neighbouring native plant species than in the presence of a neighbouring non-native plant species. Together, these results demonstrate that invaded plant communities may accumulate additional non-native species even if direct interactions between non-natives species are negative. Put another way, invasions may be more likely to lead to more invasions, requiring more active management of ecosystems by promoting native species restoration to undermine invasive positive feedback and to assist native species recovery in invaded ecosystems. PMID:27618506

  3. 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. PMID:24268446

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

    NASA Astrophysics Data System (ADS)

    Maciel, J. C.; Andrad, P. L.; Neri, D. F. M.; Carvalho, L. B.; Cardoso, C. A.; Calazans, G. M. T.; Albino Aguiar, J.; Silva, M. P. C.

    2012-04-01

    Magnetic levan was synthesized by co-precipitating D-fructofuranosyl homopolysaccharide with a solution containing Fe2+ and Fe3+ in alkaline conditions at 100 °C. The magnetic levan particles were characterized by scanning electron microscopy (SEM), magnetization measurements, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Afterwards, magnetic levan particles were functionalized by NaIO4 oxidation and used as matrices for trypsin covalent immobilization. Magnetite and magnetic levan particles were both heterogeneous in shape and levan-magnetite presented bigger sizes compared to magnetite according to SEM images. Magnetic levan particles exhibited a magnetization 10 times lower as compared to magnetite ones, probably, due to the coating layer. XRD diffractogram showed that magnetite is the dominant phase in the magnetic levan. Infrared spectroscopy showed characteristics absorption bands of levan and magnetite (O-H, C-O-C and Fe-O bonds). The immobilized trypsin derivative was reused 10 times and lost 16% of its initial specific activity only. Therefore, these magnetic levan particles can be proposed as an alternative matrices for enzyme immobilization.

  5. Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions.

    PubMed

    Avanzo, Petra; Sabotic, Jerica; Anzlovar, Sabina; Popovic, Tatjana; Leonardi, Adrijana; Pain, Roger H; Kos, Janko; Brzin, Joze

    2009-12-01

    We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K(i) in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family. PMID:19696108

  6. Tannins, trypsin inhibitors and lectin cytotoxicity in tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans.

    PubMed

    De Mejia, Elvira Gonzalez; Del Carmen Valadez-Vega, Maria; Reynoso-Camacho, Rosalia; Loarca-Pina, Guadalupe

    2005-09-01

    This study compared the levels of antinutritional components and cytotoxic effect of extracts, from tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Antinutritional factors were evaluated by determining their effect on the viability of epithelial cells isolated from rat small intestine. The protein and carbohydrates content were similar in all the genotypes studied (20 and 60%, respectively). Common beans presented higher content of trypsin inhibitors, tannins and lectins than tepary beans. There was not a significant correlation between tannins and cooking time. However, water absorption and cooking time correlated significantly (p < 0.05). Considerable variation was observed in lectin activity (1302-18161 Ul/mg) of extracts from different beans. Tannins, lectins, trypsin inhibitors and fat content differed between bean varieties whereas protein content was similar. The percent cellularity on rat epithelial cells was significantly different among protein extracts from different bean cultivars and ranged between 53.5% and 87.4% (p < 0.05). These results suggest that the incorporation of tepary beans in the diet would not alter the current nutritional contribution of common beans or introduce adverse toxic effects. The agronomic characteristics of tepary beans make them attractive for cultivation. However, the harder to cook phenomenon may be a limiting factor that needs further consideration. PMID:16187017

  7. Lunatusin, a trypsin-stable antimicrobial peptide from lima beans (Phaseolus lunatus L.).

    PubMed

    Wong, Jack Ho; Ng, Tzi Bun

    2005-11-01

    An anti-fungal peptide designated as lunatusin, with a molecular mass around 7kDa, was purified from the seeds of Chinese lima bean (Phaseolus lunatus L.). The peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. Lunatusin exerted an anti-fungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola and Botrytis cinerea, and an antibacterial action on, Bacillus megaterium, Bacillus subtilis, Proteus vulgaris and Mycobacterium phlei. It also inhibited proliferation in the breast cancer cell line MCF-7. Lunatusin reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its anti-fungal activity was retained after incubation with trypsin. Lunatusin elicited a mitogenic response from mouse splenocytes. PMID:16269344

  8. Hui Malama O Ke Kai: A Positive Prevention-Based Youth Development Program Based on Native Hawaiian Values and Activities

    ERIC Educational Resources Information Center

    Hishinuma, Earl S.; Chang, Janice Y.; Sy, Angela; Greaney, Malia F.; Morris, Katherine A.; Scronce, Ami C.; Rehuher, Davis; Nishimura, Stephanie T.

    2009-01-01

    Evaluation of after-school programs that are culturally and place-based and promote positive youth development among minority and indigenous youths has not been widely published. The present evaluation is the first of its kind of an after-school, youth-risk prevention program called Hui Malama O Ke Kai (HMK), that emphasizes Native Hawaiian values…

  9. 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

    ... service during World War II. 329.5 Section 329.5 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY... DUTY SERVICE IN THE UNITED STATES ARMED FORCES DURING SPECIFIED PERIODS OF HOSTILITIES § 329.5 Natives... Philippines; (2) Police clearance for any place of residence for more than six months in the previous 5...

  10. Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

    PubMed

    Liang, Guyan; Bowen, J Phillip

    2016-01-01

    There has been a revolution in the development of effective, small-molecule anticoagulants and antiplatelet agents. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor VIIa, factor Xa, and β-tryptase with indications ranging from thrombosis and inflammation to asthma and chronic obstructive pulmonary disease (COPD). Trypsin-like serine proteases exhibit a highly similar tertiary folding pattern, especially for the region near the substrate binding pocket that includes the conserved catalytic triad consisting of histidine 57, aspartic acid 102, and serine 195. A rich collection of X-ray structures for many trypsin-like serine proteases is available, which greatly facilitated the optimization of small organic inhibitors as therapeutic agents. The present review surveyed those inhibitors disclosed in peer-reviewed scientific journals and patent publications with a special focus on structural features and protein-inhibitor interactions that implicated the inhibitor optimization process. The role played by the residue 190 of trypsin-like serine proteases is critical. While many inhibitors without a basic group have progressed into the clinic for ones with alanine 190, the task for those with serine 190 remains extremely challenging, if not impossible. In addition to warfarin, heparin, and low molecular weight heparins (LMWHs), treatment options have expanded with the development and approval of the new oral anticoagulants (NOACs). The NOACs are superior to vitamin K antagonists in terms of rapid onset, pharmacokinetics, drug/food interactions, and regular coagulation monitoring; but one serious drawback is the lack of an effective antidote at this time. Apixaban (Eliquis), rivaroxaban (Xarelto), and edoxaban (Savaysa) are the new Xa inhibitors that have been recently approved by the U.S. FDA and are in current clinical practice. These drugs bind to the active site of factor Xa (f

  11. Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

    PubMed

    Ghale, Garima; Kuhnert, Nikolai; Nau, Werner M

    2012-03-01

    A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases. PMID:22545408

  12. 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

  13. Differences in Overweight and Obesity among Children from Migrant and Native Origin: The Role of Physical Activity, Dietary Intake, and Sleep Duration

    PubMed Central

    Rodenburg, Gerda; Koopmans, Gerrit

    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. PMID:26030064

  14. Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

    PubMed Central

    VanCott, T C; Mascola, J R; Kaminski, R W; Kalyanaraman, V; Hallberg, P L; Burnett, P R; Ulrich, J T; Rechtman, D J; Birx, D L

    1997-01-01

    Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates. PMID:9151820

  15. 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. PMID:26030064

  16. Quick and low cost immobilization of proteinases on polyesters: Comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation.

    PubMed

    Agyei, Dominic; Tambimuttu, Shaun; Kasargod, Bhuvana; Gao, Yuan; He, Lizhong

    2014-10-20

    Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40-70 °C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20 °C or in a buffer at 4 °C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes. PMID:25128611

  17. Trypsin-Ligand Binding Free Energy Calculation with AMOEBA

    PubMed Central

    Shi, Yue; Jiao, Dian; Schnieders, Michael J.; Ren, Pengyu

    2010-01-01

    The binding free energies of several benzamidine-like inhibitors to trypsin were examined using a polarizable potential. All the computed binding free energies are in good agreement with the experimental data. From free energy decomposition, electrostatic interaction was found to be the driving force for the binding. Structural analysis shows that the ligands form hydrogen bonds with residues and water molecules nearby in a competitive fashion. The dependence of binding free energy on molecular dipole moment and polarizability was also studied. While the binding free energy is independent on the dipole moment, it shows a negative correlation with the polarizability. PMID:19965178

  18. Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

    PubMed

    Gottfried, D S; Haas, E

    1992-12-15

    To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (Förster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins. PMID:1281424

  19. Comparative study of the binding of Trypsin with bifendate and analogs by spectrofluorimetry

    NASA Astrophysics Data System (ADS)

    Li, Hua; Pu, Juanjuan; Wang, Yi; Liu, Chuang; Yu, Jie; Li, Tao; Wang, Ruiqiang

    2013-11-01

    The interactions between Trypsin and bifendate (DDB) or analogs (I, II and III) were investigated by fluorescence, UV-visible absorption, resonance light scattering, synchronous fluorescence and 3D spectroscopy under mimic physiological conditions. The results revealed that DDB and analogs caused the fluorescence quenching of Trypsin by the formation of DDB/I/II/III-Trypsin complex. The quenching and energy transfer mechanisms were discussed. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that DDB was the stronger quencher and bound to Trypsin with higher affinity than other three analogs.

  20. The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

    PubMed

    Negreiros, A N; Carvalho, M M; Xavier Filho, J; Blanco-Labra, A; Shewry, P R; Richardson, M

    1991-01-01

    The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin. PMID:1367792

  1. Carboxyethylester-polyrotaxanes as a new calcium chelating polymer: synthesis, calcium binding and mechanism of trypsin inhibition.

    PubMed

    Ooya, Tooru; Eguchi, Masaru; Ozaki, Atsushi; Yui, Nobuhiko

    2002-08-21

    A carboxyethylester-polyrotaxane was synthesized as a novel calcium chelating polymer in the field of oral drug delivery and characterized in terms of mechanism of trypsin inhibition. Here, carboxyethylester (CEE) groups are introduced to all the primary hydroxyl groups in alpha-cyclodextrins (alpha-CDs), which are threaded onto a poly(ethylene glycol) chain capped with bulky end-groups (polyrotaxane). The solubility of the CEE-polyrotaxane in physiological conditions increased with pH, indicating ionization-related solubility similar to conventional polyacrylates. The ability of calcium (Ca2+) chelation was found to increase in the order of poly(acrylic acid) (PAA)>CEE-polyrotaxanez.Gt;CEE-alpha-CD, suggesting that the increased density of carboxyl groups enhances the Ca2+ chelating ability. The activity of trypsin was inhibited by these compounds in the same order of the calcium chelation. However, the inhibitory effect of CEE-polyrotaxane was reduced by adding excess Ca2+ without precipitation that was observed in the presence of PAA. Such the reduced inhibition and precipitation by CEE-alpha-CD was not observed. Therefore, the inhibitory effect of CEE-polyrotaxane is due to Ca2+ chelation from trypsin without non-specific interaction. PMID:12176224

  2. 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.

  3. [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. PMID:18672684

  4. 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

  5. Anti-inflammatory Activity of the Invasive Neophyte Polygonum Cuspidatum Sieb. and Zucc. (Polygonaceae) and the Chemical Comparison of the Invasive and Native Varieties with regard to Resveratrol.

    PubMed

    Fan, Peihong; Zhang, Tao; Hostettmann, Kurt

    2013-07-01

    Polygonum cuspidatum Sieb. and Zucc. has been traditionally used as a member of many anti-inflammatory polyherbal formulations, but is now a widespread invasive neophyte in Europe and America. To discuss if the invasive variety is chemically identical to the native one in traditional medicine, the different constituents of the invasive variety compared to the native variety were isolated and their anti-inflammatory activity was tested. Resveratroloside and catechin-(4α→8)-catechin, the newly found constituents in the invasive variety, have similar nitric oxide (NO) inhibition potency as that of piceid (the major constituent of P. cuspidatum), but the newly found major constituent, i.e., piceatannol glucoside, showed no apparent effect. On the other hand, as a marker, the total content of resveratrol in the methanol root extract after glucosidase hydrolysis was measured and compared between the invasive and native varieties. The total content of resveratrol measured in the root extracts of the Swiss sample was about 2.5 times less than that of the Chinese one. This study brings attention to the point that when the invasive variety of P. cuspidatum is used in traditional medicine, the chemical difference should be kept in mind. PMID:24716176

  6. Anti-inflammatory Activity of the Invasive Neophyte Polygonum Cuspidatum Sieb. and Zucc. (Polygonaceae) and the Chemical Comparison of the Invasive and Native Varieties with regard to Resveratrol

    PubMed Central

    Fan, Peihong; Zhang, Tao; Hostettmann, Kurt

    2013-01-01

    Polygonum cuspidatum Sieb. and Zucc. has been traditionally used as a member of many anti-inflammatory polyherbal formulations, but is now a widespread invasive neophyte in Europe and America. To discuss if the invasive variety is chemically identical to the native one in traditional medicine, the different constituents of the invasive variety compared to the native variety were isolated and their anti-inflammatory activity was tested. Resveratroloside and catechin-(4α→8)-catechin, the newly found constituents in the invasive variety, have similar nitric oxide (NO) inhibition potency as that of piceid (the major constituent of P. cuspidatum), but the newly found major constituent, i.e., piceatannol glucoside, showed no apparent effect. On the other hand, as a marker, the total content of resveratrol in the methanol root extract after glucosidase hydrolysis was measured and compared between the invasive and native varieties. The total content of resveratrol measured in the root extracts of the Swiss sample was about 2.5 times less than that of the Chinese one. This study brings attention to the point that when the invasive variety of P. cuspidatum is used in traditional medicine, the chemical difference should be kept in mind. PMID:24716176

  7. Relationship between Single Nucleotide Polymorphisms in the Peroxisome Proliferator-Activated Receptor Gamma Gene and Fatty Acid Composition in Korean Native Cattle.

    PubMed

    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

    2016-02-01

    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

  8. [Proteolytic enzymes from Streptomyces fradiae: a metalloendopeptidase, subtilisin-like, and trypsin-like proteinases].

    PubMed

    Bormatova, M E; Ivanova, N M; Iusupova, M P; Voiushina, T L; Surova, I A; Chestukhina, G G; Stepanov, V M

    1996-02-01

    Three proteolytic enzymes-the metalloproteinase, SFMP, and two serine proteinases, SFSP and SFTP-have been isolated and purified from the culture fluid of Streptomyces fradiae using chromatography on bacitracin-silochrome, bacitracin-Sepharose, DEAE-cellulose and fractionation by ammonium sulfate. Study of physico-chemical and functional properties of the enzymes and structural analysis revealed that SFMP is a cysteine-containing metalloendopeptidase with M(r) of 36 kDa, has a peak activity for synthetic substrates at pH 7.0-7.5 and at 60-65 degrees C and is stable at pH 7.0-9.0. The serine proteinase SFSP is related to subtilisin-like enzymes, has a M(r) of 29 kDa and a pH optimum at 7.5-8.5 at temperature up to 50 degrees C. The proteinase is stable at pH 4.0-9.0 and retains 30% of its activity at 70 degrees C. The other serine proteinase, SFTP, has a M(r) of 26 kDa and is related to trypsin-like enzymes. Its activity for synthetic substrates of trypsin is maximal at pH 6.8-8.8 at 50 degrees C. The enzyme is stable at pH 4.5-8.5 and at temperature below 50 degrees C. It has been shown that Streptomyces fradiae, like Streptomyces griseus and other Streptomycetes, possesses an ability to secrete serine proteinases (SFSP and SFTP) related to two evolutionally distinct families of serine proteinases, i.e., subtilisin and chymotrypsin families. SFMP and SFSP have been isolated and characterized for the first time. PMID:8717499

  9. Size-dependent optical properties of bio-compatible ZnS:Mn nanocrystals and their application in the immobilisation of trypsin

    NASA Astrophysics Data System (ADS)

    Sajimol Augustine, M.; Manzur Ali, P. P.; Sapna, K.; Elyas, K. K.; Jayalekshmi, S.

    2013-05-01

    Chitosan capped zinc sulphide nanocrystals doped with manganese (ZnS:Mn) have been synthesised by chemical capping co-precipitation method and structurally characterised by XRD, TEM and EDXS techniques. The dependence of optical properties on the size of these bio-compatible ZnS:Mn nanocrystals was investigated by UV/Vis and photoluminescence (PL) spectroscopic techniques in aqueous solvents. A variation in molar concentration of the precursor, sodium sulphide, from 0.125 to 0.01 mol L-1 is accompanied by a decrease in particle size. The excitonic peak in the UV/Vis spectra is found to be blue shifted with a decrease in size of the nanocrystals due to confinement effects. In the present study, trypsin was immobilised onto ZnS:Mn nanocrystals using glutaraldehyde (GA) as cross-linker, which was confirmed by photoluminescence (PL) and Fourier transform infrared (FTIR) spectroscopic studies. Results indicate that the activity of trypsin, immobilised onto chitosan modified nanocrystals, has improved upon cross-linking, which suggests that the immobilised trypsin has become more stable and active. This work highlights the prospects of potential applications of immobilised trypsin in therapeutic and diagnostic fields.

  10. 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; School of Medical Sciences and Bosch Institute, University of Sydney, NSW 2006

    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.

  11. Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes

    PubMed Central

    Saleh, Sohag N; Albert, Anthony P; Large, William A

    2009-01-01

    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 ETA or ETB 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 ETA and ETB receptor stimulation activate this conductance. Stimulation of both ETA and ETB 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. ETA 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 (PIP3), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2). Moreover, exogenous application of diC8-PIP3 stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ETA receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP3. In contrast, ETB 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 (PIP2) antibodies and high concentrations of wortmannin (20 μm) which depleted tissue PIP2 levels. In

  12. 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. PMID:25192870

  13. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Liquid crystalline trypsin, Peru balsam, castor... NEW ANIMAL DRUGS § 524.2620 Liquid crystalline trypsin, Peru balsam, castor oil. (a)(1) Specifications. The drug is a liquid for direct application or an aerosol preparation formulated so that each...

  14. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Liquid crystalline trypsin, Peru balsam, castor... NEW ANIMAL DRUGS § 524.2620 Liquid crystalline trypsin, Peru balsam, castor oil. (a)(1) Specifications. The drug is a liquid for direct application or an aerosol preparation formulated so that each...

  15. 21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Liquid crystalline trypsin, Peru balsam, castor... NEW ANIMAL DRUGS § 524.2620 Liquid crystalline trypsin, Peru balsam, castor oil. (a)(1) Specifications. The drug is a liquid for direct application or an aerosol preparation formulated so that each...

  16. Cloning and transcription profiling of trypsin in Aedes taeniorhynchus (Diptera: Culicdae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA of a trypsin gene from Aedes (Ochlerotatus) taeniorhynchus (Weidemann) was cloned and sequenced. THe full-length mRNA sequence (874 bp_ for trypsin from Ae.taeniorhynchus (AetTryp_ was obtained which encodes an open reading frame of 717 bp (i.e., 239 aa). To detect whether AetTryp is develo...

  17. Antifungal traits of a 14 kDa maize kernel trypsin inhibitor protein in transgenic cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic cotton plants expressing the maize kernel trypsin inhibitor (TI) protein were produced and evaluated for antifungal traits. This 14 kD trypsin inhibitor protein has been previously associated with resistance to aflatoxin-producing fungus Aspergillus flavus. Successful transformation of ...

  18. Effect of nitrate, acetate and hydrogen on native perchlorate-reducing microbial communities and their activity in vadose soil

    PubMed Central

    Nozawa-Inoue, Mamie; Jien, Mercy; Yang, Kun; Rolston, Dennis E.; Hristova, Krassimira R.; Scow, Kate M.

    2011-01-01

    Effect of nitrate, acetate and hydrogen on native perchlorate-reducing bacteria (PRB) was examined by conducting microcosm tests using vadose soil collected from a perchlorate-contaminated site. The rate of perchlorate reduction was enhanced by hydrogen amendment and inhibited by acetate amendment, compared to unamendment. Nitrate was reduced before perchlorate in all amendments. In hydrogen-amended and unamended soils, nitrate delayed perchlorate reduction, suggesting the PRB preferentially use nitrate as an electron acceptor. In contrast, nitrate eliminated the inhibitory effect of acetate amendment on perchlorate reduction and increased the rate and the extent, possibly because the preceding nitrate reduction/denitrification decreased the acetate concentration which was inhibitory to the native PRB. In hydrogen-amended and unamended soils, perchlorate reductase gene (pcrA) copies, representing PRB densities, increased with either perchlorate or nitrate reduction, suggesting either perchlorate or nitrate stimulates growth of the PRB. In contrast, in acetate-amended soil pcrA increased only when perchlorate was depleted: a large portion of the PRB may have not utilized nitrate in this amendment. Nitrate addition did not alter the distribution of the dominant pcrA clones in hydrogen-amended soil, likely because of the functional redundancy of PRB as nitrate-reducers/denitrifiers, whereas acetate selected different pcrA clones from those with hydrogen amendment. PMID:21284679

  19. 5-HT1A receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

    PubMed Central

    Watson, J; Collin, L; Ho, M; Riley, G; Scott, C; Selkirk, J V; Price, G W

    2000-01-01

    It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT1A receptors. Saturation studies using [3H]-8-OH-DPAT and [3H]-MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5-HT1A receptors and rat cortex. In recombinant cells, [3H]-MPPF labelled 3–4 fold more sites than [3H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [3H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT1A receptors but did not bind to native tissue 5-HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5-HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. Receptor agonists inhibited [3H]-MPPF binding from recombinant 5-HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]-8-OH-DPAT and [3H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]-MPPF and [3H]-8-OH-DPAT in a monophasic manner. Functional evaluation of compounds, using [35S]-GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. [3H]-8-OH-DPAT : [3H]-MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]-8-OH-DPAT : [3H]-spiperone pKi difference with functional intrinsic activity (r=0.96). Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT1A receptors in which

  20. [Development of a new soybean variety with null trypsin inhibitor and lipoxygenase 2.3 genes--zhonghuang 16 and its cultivation practices].

    PubMed

    Han, Fen-Xia; Ding, An-Lin; Sun, Jun-Ming

    2002-12-01

    Soybean protein is a kind of high-quality protein composed of balanced amino acids, which contains all kinds of amino acid, especially 8 amino acids necessary for human, but also contains some components that are not good for human and affect food quality, such as Trypsin inhibitor and Lipoxygenase. Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, a new soybean variety Zhonghuang 16 (originally name as Zhongzuo 96-952) was developed by Institute of Crop Breeding and Cultivation Chinese Academy of Agricultural Sciences through years of biochemical marker assistant selection for null trypsin inhibitor by Native-polyacrylamid gel electrophoresis (Native-PAGE) and null lipoxygenase by means of isoelectric focusing-polyacrylamid gel electrophoresis (IEF-PAGE) in the hybrid progenies of "ti15176" (Female parent)--a high-yielding, mosaic virus resistant and null trypsin inhibitor line and "Century-2.3" (Male parent)--a null lipoxygenase near isogene line of an elite American variety "Century". This variety was subjected to Beijing regional trial for summer-sowing soybean during 1999-2000, and to Beijing demonstration test in 2001. In 2002, it was passed the examination and approval by the Beijing Committee of Crop Variety Examination and Approval because of its outstanding characteristics such as high and stable yielding, good quality (high protein and fat content, high protein content and good protein quality-null Ti and Lox2.3), disease resistant and good general character. It is the first new soybean variety with null Ti and Lox2.3 genes in our country. In this paper, the development process and cultivation of Zhonghuang 16 were described. PMID:12693103

  1. 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 Central

    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

    2013-01-01

    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. PMID:23381957

  2. 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.

  3. Cytometric analysis of retinopathies in retinal trypsin digests

    NASA Astrophysics Data System (ADS)

    Ghanian, Zahra; Staniszewski, Kevin; Sorenson, Christine M.; Sheibani, Nader; Ranji, Mahsa

    2014-03-01

    The objective of this work was to design an automated image cytometry tool for determination of various retinal vascular parameters including extraction of features that are relevant to postnatal retinal vascular development, and the progression of diabetic retinopathy. To confirm the utility and accuracy of the software, retinal trypsin digest from TSP1-/- and diabetic Akita/+; TSP1-/- mice were analyzed. TSP1 is a critical inhibitor of development of retinopathies and lack of TSP1 exacerbates progression of early diabetic retinopathies. Loss of vascular cells of and gain more acellular capillaries as two major signs of diabetic retinopathies were used to classify a retina as normal or injured. This software allows quantification and high throughput assessment of retinopathy changes associated with diabetes.

  4. 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.

  5. Comparative analysis of expression profiling of the trypsin and chymotrypsin genes from Lepidoptera species with different levels of sensitivity to soybean peptidase inhibitors.

    PubMed

    Souza, Thais P; Dias, Renata O; Castelhano, Elaine C; Brandão, Marcelo M; Moura, Daniel S; Silva-Filho, Marcio C

    2016-01-01

    Peptidase inhibitors (PIs) are essential proteins involved in plant resistance to herbivorous insects, yet many insect species are able to escape the negative effects of these molecules. We compared the effects of acute and chronic ingestion of soybean peptidase inhibitors (SPIs) on Spodoptera frugiperda and Diatraea saccharalis, two Lepidoptera species with different sensitivities to SPI ingestion. We analyzed the trypsin and chymotrypsin gene expression profiles in both species. Acute exposure of S. frugiperda to the inhibitors activated seven genes (SfChy5, SfChy9, SfChy19, SfChy22, SfTry6, SfTry8, and SfTry10), whereas chronic exposure activated 16 genes (SfChy2, SfChy4, SfChy5, SfChy8, SfChy9, SfChy11, SfChy12, SfChy15, SfChy17, SfChy21, SfChy22, SfTry6, SfTry8, SfTry9, SfTry10, and SfTry12). By contrast, the challenge of D. saccharalis with SPIs did not differentially induce the expression of trypsin- or chymotrypsin-encoding genes, with the exception of DsChy7. Bayesian phylogenetic analysis of S. frugiperda trypsin protein sequences revealed two gene clades: one composed of genes responsive to the SPIs and a second composed of the unresponsive genes. D. saccharalis trypsin proteins were clustered nearest to the S. frugiperda unresponsive genes. Overall, our findings support a hypothesized mechanism of resistance of Noctuidae moths to SPIs, involving gene number expansion of trypsin and chymotrypsin families and regulation of gene expression, which could also explain the variable susceptibility between S. frugiperda and D. saccharalis to these plant inhibitors. PMID:26944308

  6. Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure.

    PubMed

    Hernandez, J F; Gagnon, J; Chiche, L; Nguyen, T M; Andrieu, J P; Heitz, A; Trinh Hong, T; Pham, T T; Le Nguyen, D

    2000-05-16

    Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities. PMID:10801322

  7. 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

  8. Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

    PubMed Central

    Zhang, Xinyu; Gunner, M. R.

    2014-01-01

    Purple, photosynthetic reaction centers (RCs) from Rb. sphaeroides bacteria use UQ10 as primary (QA) and secondary (QB) electron acceptors. Many quinones reconstitute QA function, while few will act as QB. Nine quinones were tested for their ability to bind and reconstitute QA and QB function. Only ubiquinone (UQ) reconstitutes both QA and QB function in the same protein. The affinities of the non-native quinones for the QB site were determined by a competitive inhibition assay. The affinities of benzoquinones (BQ), napthoquinone (NQ) and 2-methyl-NQ for the QB site are 7±3 times weaker than for the QA site. However, di-ortho substituted NQs and anthraquinone bind tightly to the QA site (Kd ≤200 nM) and ≥1000 times more weakly to the QB site, perhaps setting a limit on the size of the site. With a low potential electron donor (2-methyl, 3-dimethylamino-1,4-Napthoquinone (Me-diMeAm-NQ)) at QA, QB reduction is 260 meV more favorable than with UQ as QA. Electron transfer from Me-diMeAm-NQ at the QA site to NQ at the QB site can be detected. In the QB site the NQ semiquinone is estimated to be ≈ 60–100 meV higher in energy than the UQ semiquinone, while in the QA site the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the QA than QB site. In contrast, the native UQ semiquinone is ≈ 60 meV lower in energy in the QB than the QA site, stabilizing forward electron transfer from QA to QB. PMID:23715773

  9. 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 Pokagon's…

  10. 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

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

    PubMed

    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

  12. THE EFFECT OF CALCIUM AND OTHER IONS ON THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN.

    PubMed

    McDonald, M R; Kunitz, M

    1941-09-20

    Crystalline trypsinogen is completely transformed into trypsin by means of trypsin in the presence of calcium salts. The process follows the course of a pure autocatalytic unimolecular reaction. In the absence of calcium salts, the autocatalytic formation of trypsin from trypsinogen is complicated by the transformation of part of the trypsinogen into an inert protein which cannot be changed into trypsin by any known means. Salts increase or decrease the rate of both reactions so that the ultimate amount of trypsin formed varies with the nature and concentration of the salt used. With equivalent concentrations of salt the percentage of trypsinogen changed into trypsin is greatest in the presence of calcium ion followed in order by strontium; magnesium and sodium; rubidium, ammonium, lithium, and potassium; caesium and barium. With the anions the largest percentage of trypsinogen transformed into trypsin was found with the acetate, sulfate, oxalate, citrate, tartrate, fluoride, and chloride ions followed in order by bromide, nitrate, and iodide. The formation of inert protein is completely suppressed by concentrations of calcium ion greater than 0.02 M. PMID:19873258

  13. Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia.

    PubMed

    Liu, Zubi; Zhu, Qiankun; Li, Juanjuan; Zhang, Gan; Jiamahate, Aerguli; Zhou, Jiayu; Liao, Hai

    2015-04-01

    A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A, R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress. PMID:25479703

  14. Structure of the dinuclear active site of urease. X-ray absorption spectroscopic study of native and 2-mercaptoethanol-inhibited bacterial and plant enzymes

    SciTech Connect

    Wang, Shengke; Scott, R.A. ); Lee, M.H.; Hausinger, R.P. ); Clark, P.A.; Wilcox, D.E. )

    1994-04-13

    The structures of the dinuclear Ni(II) active sites of urease from jack bean and Klebsiella aerogenes are compared with and without the addition of the inhibitor 2-mercaptoethanol (2-ME). No significant differences are observed by nickel K-edge X-ray absorption spectroscopy between the plant and bacterial enzymes. The Ni X-ray absorption edge spectra display an 8332-eV 1s[yields]3d peak intensity similar to that observed for five-coordinate Ni(II) compounds[sup 1] for both native and 2-ME-bound derivatives. Curve-fitting of Ni EXAFS data indicates that the average Ni(II) coordination environment in native urease can be described as Ni(imidazole)[sub x](N,O)[sub 5[minus]x], with x = 2 or 3. Addition of 2-ME results in replacement of one of the non-imidazole (N,O) ligands with (S,Cl) (most likely the thiolate sulfur of 2-ME) and results in the appearance of a new peak in the Fourier transforms that can only be fit with a Ni[center dot][center dot][center dot]Ni scattering component at a Ni-Ni distance of [approximately]3.26 [angstrom]. A structure for this 2-ME-bound dinuclear site is proposed to contain the two Ni(II) ions bridged by the thiolate sulfur of 2-ME.

  15. 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 Indians Study the Heavenly Bodies?"…

  16. 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. PMID:27542490

  17. Spectroscopic study on the interaction of Trypsin with Bicyclol and analogs

    NASA Astrophysics Data System (ADS)

    He, Wu; Dou, Huanjing; Zhang, Lu; Wang, Lvjing; Wang, Ruiyong; Chang, Junbiao

    2014-01-01

    The interactions between Trypsin and Bicyclol or analogs (Bifendate, I, II and III) were investigated by spectrophotometric methods. It was found that Bicyclol or analogs had strong ability to quench the intrinsic fluorescence of Trypsin by a static quenching procedure. The binding constants were obtained at three temperatures. The thermodynamics parameters reveal that the hydrophobic and electrostatic interactions play an important role in the interaction. Results showed that the microenvironments of tryptophan residue of Trypsin were disturbed by the analogs. Results indicated that Bifendate was the strongest quencher among five compounds.

  18. Nonlocal interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor.

    PubMed

    Ittah, V; Haas, E

    1995-04-01

    A search for the topology of the chain folding of reduced bovine pancreatic trypsin inhibitor (BPTI), in unfolded and partially folded states, was done by means of time resolved dynamic nonradiative excitation energy transfer (ET) measurements. Four double labeled BPTI derivatives were used in which the donor was attached to the N-terminal arginine residue and the acceptor was specifically attached to one of the lysine residues. The four derivatives form a series of labeled backbone segments of increasing length spanning the full lengths of the BPTI chain: 15, 26, 41, and 46 residues. The intramolecular segmental end-to-end distance (EED) distributions were determined for all the derivatives by global analysis of the decay curves of both the donor and the acceptor in the reduced state, in low (0.5 M) guanidinium chloride (GuHCl) concentrations at pH 3.6 and 2.1 (R and A states, respectively). The results show that, in the partial folding conditions of low GuHCl concentration, reduced BPTI is in a compact state, but in this state the polypeptide chain is not in a condensed statistical coil conformation. Two distinct subpopulations were found for the four intramolecular EED distributions. One subpopulation was compact, with native-like EED distribution, while the second was unfolded. The pairs of sites, residues 1 and 26 and residues 1 and 46, showed close proximity in the dominant subpopulation. These contacts form two loops (probably collapsed): one consists of the first 26 residues, and the second comprises the full length of the chain from the N- to the C-terminal segments, which is in fact made up to two shorter loops (1-26 and 27-46). The N-terminal 15 residue segment was relaxed into statistical coil-like non-native conformation, in contrast to its extended conformation in the native state. The effect of temperature in the range of 2-60 degrees C was small; the folded subpopulations were stable over this range. These results show that in BPTI the compact

  19. Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

    PubMed

    Sturm, M E; Assof, M; Fanzone, M; Martinez, C; Ganga, M A; Jofré, V; Ramirez, M L; Combina, M

    2015-08-01

    Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality. PMID:25955288

  20. Correlating the structures and activities of the resting oxidized and native intermediate states of a small laccase by paramagnetic NMR.

    PubMed

    Machczynski, Michael C; Babicz, Jeffrey T

    2016-06-01

    Multicopper oxidases (MCO) are the fastest and most efficient known catalysts of the oxygen-reduction reaction. When all four copper ions are oxidized during catalysis, the native intermediate state (NI) decays in seconds to the resting oxidized state (RO), which returns to the catalytic cycle via reduction, but at a much slower rate than NI. We report the long-lived (months at 4°C) NI state of the small laccase (SLAC) MCO and the subsequent characterization of both its RO and NI states by paramagnetic (1)H NMR. We find that the RO state of the trinuclear cluster (TNC) is best described as an isolated Type-3 dicopper site, antiferromagnetically coupled by a hydroxo group with -2J=500cm(-1). The NI state is more complicated; we develop a theoretical treatment for the case in which all three copper ions in the TNC are coupled, and find that the results are consistent with three coupling constants of -2J=300, 240, and 160cm(-1). These couplings result in a ground doublet state, a low-lying excited doublet state at 121cm(-1), and a quartet excited state at 411cm(-1), in good agreement with DFT models in which the Type-2 copper has a terminal hydroxo ligand. PMID:26918900

  1. Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.

    PubMed

    Avrutina, Olga; Schmoldt, Hans-Ulrich; Gabrijelcic-Geiger, Dusica; Le Nguyen, Dung; Sommerhoff, Christian P; Diederichsen, Ulf; Kolmar, Harald

    2005-12-01

    MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity. PMID:16336125

  2. Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin.

    PubMed

    Tomar, Sakshi; Patil, Dipak N; Datta, Manali; Tapas, Satya; Preeti; Chaudhary, Anshul; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra

    2009-11-01

    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 angstrom resolution and belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 57.1, c = 120.1 angstrom. 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

  3. Plants used in Guatemala for the treatment of protozoal infections. I. Screening of activity to bacteria, fungi and American trypanosomes of 13 native plants.

    PubMed

    Cáceres, A; López, B; González, S; Berger, I; Tada, I; Maki, J

    1998-10-01

    Extracts were prepared from 13 native plants used for the treatment of protozoal infections. Activity against bacteria and fungi was demonstrated by dilution procedures; Trypanosoma cruzi was evaluated in vitro against epimastigote and trypomastigotes and in vivo against trypomastigotes. In active extracts, toxicity was evaluated by Artemia salina nauplii, oral acute toxicity (1-5 g/kg) and oral and intraperitoneal subacute toxicity in mice (500 mg/kg). From the plants screened, six showed activity (< or = 2 mg/ml) against bacteria, three against yeasts, five against Microsporum gypseum and five against T. cruzi in vitro and/or in vivo. In vitro and in vivo activity was demonstrated by Neurolaena lobata and Solanum americanum; in vitro or in vivo activity was shown by Acalypha guatemalensis, Petiveria alliacea and Tridax procumbens. Toxicity studies showed that extracts from S. americanum are toxic to A. salina (aqueous, 160 ppm). None showed acute or oral toxicity to mice; S. americanum showed intraperitoneal subacute toxicity. PMID:9849628

  4. 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

  5. Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods.

    PubMed

    Wang, Yan-Qing; Chen, Ting-Ting; Zhang, Hong-Mei

    2010-03-01

    The interaction between bisphenol A (BPA) and lysozyme (or trypsin) was investigated by UV-vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. BPA effectively quenched the intrinsic fluorescence of lysozyme and trypsin via static quenching. H-bonds and van der Waals interactions played a major role in stabilizing the BPA-proteinase complex. The distance r between donor and acceptor was obtained to be 1.65 and 2.26 nm for BPA-lysozyme and BPA-trypsin complexes, respectively. The effect of BPA on the conformation of lysozyme and trypsin was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra. PMID:20093070

  6. Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Qing; Chen, Ting-Ting; Zhang, Hong-Mei

    2010-03-01

    The interaction between bisphenol A (BPA) and lysozyme (or trypsin) was investigated by UV-vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. BPA effectively quenched the intrinsic fluorescence of lysozyme and trypsin via static quenching. H-bonds and van der Waals interactions played a major role in stabilizing the BPA-proteinase complex. The distance r between donor and acceptor was obtained to be 1.65 and 2.26 nm for BPA-lysozyme and BPA-trypsin complexes, respectively. The effect of BPA on the conformation of lysozyme and trypsin was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra.

  7. Inhibition of Sindbis virus maturation after treatment of infected cells with trypsin.

    PubMed Central

    Adams, R H; Brown, D T

    1982-01-01

    Brief treatment of Sindbis virus-infected BHK-21 or Vero cells with low concentrations of trypsin irreversibly blocked further production of progeny virions after removal of the enzyme. The inhibitory effects of the trypsin treatment could only be demonstrated in cells in which virus infection was established; optimal inhibition occurred at ca. 3 h postinfection. Production of virus structural proteins PE2, E1, and C occurred at normal levels in inhibited cells. PE2 and E1 were also transported to the cell plasma membrane during inhibition; however, PE2 was not cleaved to E2, and little capsid protein became membrane associated relative to control cells. Although trypsin treatment had no effect on Sindbis protein synthesis, the production of both 26S and 42S RNA was greatly reduced. Similar trypsin treatment of BHK cells infected with vesicular stomatitis virus had no detectable effect on the course of virus infection. Images PMID:6281478

  8. Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

    PubMed

    Mosolov, V V; Fedurkina, N V; Valueva, T A

    1978-01-12

    An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties. PMID:413581

  9. 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. PMID:24579519

  10. 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…

  11. Clonidine displacement from type 1 imidazoline receptor by p-aminobenzamidine, the prototype of trypsin-like serine protease inhibitors.

    PubMed

    Pallottini, Valentina; Marino, Maria; Ascenzi, Paolo

    2002-11-01

    p-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I1-R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [3H]clonidine displacement from I1-R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [3H]clonidine from I1-R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control. PMID:12587981

  12. A sensitive and label-free trypsin colorimetric sensor with cytochrome c as a substrate.

    PubMed

    Zhang, Lufeng; Du, Jianxiu

    2016-05-15

    The development of simple and sensitive methods for protease sensing plays important roles in clinical diagnostics and drug development. Here a simple, rapid, label-free, and sensitive trypsin colorimetric sensor was developed by employing cytochrome c (cyt c) as an enzyme substrate and 3,3´,5,5´-tetramethylbenzidine (TMB) as a chromogenic reagent. It was found that cyt c hardly catalyzes H2O2-mediated TMB oxidation to produce a blue solution. But the hydrolysate of cyt c by trypsin displays an intense catalytic effect on the aforementioned reaction, resulting in the formation of a blue solution immediately. The detection process allows visually perceiving as low as 50 ng/mL trypsin with the naked eyes. With the aid of a spectrophotometer, the absorbance at 652 nm was proportional to the concentration of trypsin in the range from 5.0 ng/mL to 2.0 μg/mL with a detection limit of 4.5 ng/mL. The sensor showed better precision with relative standard deviation of 2.5% and 1.7% for eleven repetitive measurements of 50.0 ng/mL and 1.0 μg/mL trypsin solution, respectively. The procedure has been successfully applied to the determination of trypsin in human urines and for inhibitor screening, demonstrating its potential application in clinic diagnosis and drug development. PMID:26724537

  13. Aedes aegypti midgut early trypsin is post-transcriptionally regulated by blood feeding.

    PubMed

    Noriega, F G; Pennington, J E; Barillas-Mury, C; Wang, X Y; Wells, M A

    1996-02-01

    Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal. PMID:8630532

  14. 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…

  15. 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…

  16. 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…

  17. 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

  18. Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Rodríguez-Casariego, Javier; Fraga, Iliana; Carrillo, Olimpia; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2012-03-01

    The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency. PMID:22323208

  19. Synthetic covalently linked dimeric form of H2 relaxin retains native RXFP1 activity and has improved in vitro serum stability.

    PubMed

    Nair, Vinojini B; Bathgate, Ross A D; Separovic, Frances; Samuel, Chrishan S; Hossain, Mohammed Akhter; Wade, John D

    2015-01-01

    Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and "click" chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin. PMID:25685807

  20. Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has Improved In Vitro Serum Stability

    PubMed Central

    Nair, Vinojini B.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed Akhter; Wade, John D.

    2015-01-01

    Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin. PMID:25685807

  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. 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

  3. Digestive Duet: Midgut Digestive Proteinases of Manduca sexta Ingesting Nicotiana attenuata with Manipulated Trypsin Proteinase Inhibitor Expression

    PubMed Central

    Zavala, Jorge A.; Giri, Ashok P.; Jongsma, Maarten A.; Baldwin, Ian T.

    2008-01-01

    Background The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants. Methodology/ Principal Findings Second and third instars larvae that fed on NaTPI-producing (WT) genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content. Conclusions/ Significance Although larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance. PMID:18431489

  4. 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)

  5. 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.

  6. Native oligodeoxynucleotides specifically active against human immunodeficiency virus type 1 in vitro: a G-quartet-driven effect?

    PubMed

    Tondelli, L; Colonna, F P; Garbesi, A; Zanella, S; Marongiu, M E; Corrias, S; Loi, A G; La Colla, P

    1996-09-01

    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

  7. 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

  8. 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. PMID:26090459

  9. Screening Active Compounds from Garcinia Species Native to China Reveals Novel Compounds Targeting the STAT/JAK Signaling Pathway

    PubMed Central

    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. PMID:26090459

  10. Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

    PubMed

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

    2016-12-15

    Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. PMID:27451220

  11. Distribution of Genes Encoding the Trypsin-Dependent Lantibiotic Ruminococcin A among Bacteria Isolated from Human Fecal Microbiota

    PubMed Central

    Marcille, F.; Gomez, A.; Joubert, P.; Ladiré, M.; Veau, G.; Clara, A.; Gavini, F.; Willems, A.; Fons, M.

    2002-01-01

    Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota. PMID:12089024

  12. Inhibition of serine proteinases plasmin, trypsin, subtilisin A, cathepsin G, and elastase by LEKTI: a kinetic analysis.

    PubMed

    Mitsudo, Kenji; Jayakumar, Arumugam; Henderson, Ying; Frederick, Mitchell J; Kang, Ya'an; Wang, Mary; El-Naggar, Adel K; Clayman, Gary L

    2003-04-01

    The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K(i)) of 27 +/- 5, 49 +/- 3, 67 +/- 6, 317 +/-36, and 849 +/- 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases. PMID:12667078

  13. Relative importance of phytohemagglutinin (lectin) and trypsin-chymotrypsin inhibitor on bean (Phaseolus vulgaris L) protein absorption and utilization by the rat.

    PubMed

    Carvalho, M R; Sgarbieri, V C

    1998-10-01

    The main objective of this work was to perform a comparative study of the antinutritional and/or toxic properties of phytohemagglutinin and trypsin-chymotrypsin inhibitor extracted from the seed of a commercial cultivar of edible bean used in Brazil. Bean proteins were extracted in acidic salt solution and fractionated by dialysis and centrifugation, then freeze-dried. The total freeze-dried bean extract and the globulin or albumin protein fraction were resuspended in distilled water and heated (100 degrees C, 30 min) for inactivation of hemagglutinin. Diets were prepared with unheated bean protein fractions and heated ones (100% trypsin inhibitor activity, but 0% phytohemagglutinin activity). As a result, the inhibition of growth and poor dietary protein utilization were observed in rats fed diets containing unheated bean protein fractions, but not in rats fed diets containing heated fractions. It was thus assumed that phytohemagglutinin is the main antinutritional and toxic factor that in dry bean (Phaseolus) protein and that trypsin inhibitor (Bowman-Birk type) did not interfere with rat growth. PMID:9919488

  14. Library Services for Indian Tribes and Hawaiian Natives Program: Review of Program Activities, 1990. Title IV, Library Services and Construction Act.

    ERIC Educational Resources Information Center

    Fine, Beth, Comp.; Woolen, Viola, Comp.

    THe Library Services for Indian Tribes and Hawaiian natives Program, Title IV of the Library Services and Construction Act (LSCA), supports public library services to Indians and Hawaiian Natives. Two types of grants--Basic Grants and Special Projects--encourage the development and improvement of public library services through selected project…

  15. Primary structures of four trypsin inhibitor E homologs from venom of Dendroaspis angusticeps: structure-function comparisons with other dendrotoxin homologs.

    PubMed

    Sigle, Randy; Hackett, Murray; Aird, Steven D

    2002-03-01

    Four trypsin inhibitor homologs, the first known from Dendroaspis angusticeps venom, were characterized using a combination of gel filtration, cation exchange, reverse-phase liquid chromatography, Edman degradation and mass spectrometry. The four toxins comprise two 57 residue and two 59 residue isoforms. The long toxins possess a Lys-Gln N-terminal extension lacked by the short toxins. The only other structural difference is an Arg/His replacement at position 55. The long Arg55 variant is identical to trypsin inhibitor E from the venom of Dendroaspis polylepis. The name epsilon-dendrotoxin is suggested so as to follow the nomenclature of Benishin, C.G., Sorensen, R.G., Brown, W.E., Krueger, B.K., Blaustein, M.P., 1988. Four polypeptide components of green mamba venom selectively block certain potassium channels in rat brain synaptosomes. Mol. Pharmacol. 34, 152-159. Among snake venom protease inhibitors, the epsilon-dendrotoxins are structurally most like the delta-dendrotoxins, with which they share only 64% of their residues. In addition, the epsilon-dendrotoxins display hydropathy profiles more like those of the alpha- and delta-dendrotoxins, than those of the trypsin inhibitors from snake venoms. Given the strong protease inhibitory activity of trypsin inhibitor E and the recently demonstrated weak K(+) channel inhibitory activity of two of these variants (Tytgat, J., Vandenberghe, I., Ulens, C., Van Beeumen, J., 2001. New polypeptide components purified from mamba venom. FEBS Lett. 491, 217-221), the epsilon-dendrotoxins represent structural and functional intermediates between the facilitatory toxins and the protease inhibitors. PMID:11711127

  16. Trypsin inhibitor from Moringa oleifera flowers interferes with survival and development of Aedes aegypti larvae and kills bacteria inhabitant of larvae midgut.

    PubMed

    Pontual, Emmanuel Viana; de Lima Santos, Nataly Diniz; de Moura, Maiara Celine; Coelho, Luana Cassandra Breitenbach Barroso; do Amaral Ferraz Navarro, Daniela Maria; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes

    2014-02-01

    Moringa oleifera flower extract, with trypsin inhibitor activity, is a larvicidal agent on Aedes aegypti. This work reports the isolation of trypsin inhibitor (M. oleifera flower trypsin inhibitor (MoFTI)) and its effect on A. aegypti egg hatching, viability of newly hatched larvae, survival of pupae, and growth of inhabitant bacteria from midgut of fourth-instar larvae (L4). MoFTI (K i, 2.4 μM), isolated by affinity chromatography on trypsin-agarose column, was an 18.2 kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Flower extract (at concentrations of 8.5-17.0 mg/mL) reduced egg hatchability while MoFTI (0.05-0.5 mg/mL) did not affect the hatching rate. Mortality of newly hatched larvae ranged from 3.5 to 19.1 % in the presence of the extract (4.0-17.0 mg/mL) and was also promoted by MoFTI (LC50, 0.3 mg/mL). After 72 h, larvae incubated with extract at 13.0 and 17.0 mg/mL were at stages L2 and L1, respectively, while in control they reached L3 instar. In the presence of MoFTI, at all concentrations tested, the larvae did not pass the first instar. Flower extract and MoFTI did not interfere on pupae survival. The extract and MoFTI inhibited the growth of L4 gut bacteria (minimum inhibitory concentrations of 3.47 and 0.031 mg/mL, respectively) but only the inhibitor showed bactericide effect (minimum bactericidal concentration of 1.0 mg/mL). The findings reported herein indicate that MoFTI constitutes a larvicidal principle from M. oleifera flowers against A. aegypti newly hatched larvae and is an antibacterial agent active against the microbiota from L4 gut. PMID:24271154

  17. 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

  18. Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

    PubMed Central

    Zhan, Changyou; Varney, Kristen; Yuan, Weirong; Zhao, Le; Lu, Wuyuan

    2012-01-01

    The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal “lid” (residues 1–24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1–109)MDM2 and its Ser17-phosphorylated analog (1–109)MDM2 pS17 as well as (1–109)MDM2 S17D and (25–109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1–109)MDM2 by the (15–29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation. PMID:22444248

  19. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    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

  20. 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. PMID:24050300

  1. 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…

  2. Determination of phenolic compounds content and antioxidant activity in skin, pulp, seed, cane and leaf of five native grape cultivars in West Azerbaijan province, Iran.

    PubMed

    Farhadi, Khalil; Esmaeilzadeh, Forough; Hatami, Mehdi; Forough, Mehrdad; Molaie, Rahim

    2016-05-15

    In the present work, the phenolic compounds content and antioxidant activity in the skin, pulp, seed, cane and leaf of one international (Muscat) and five native (Hosseini, Ghara Shira, Agh Shani, Ghara Shani and Ghara Ghandome) grape cultivated in West Azerbaijan, Iran were investigated. Ghara Shani grape skin was found to contain the highest content of total phenolic and anthocyanin and cane of Ghara Shani contains the highest amount of flavonoid. A remarkable DPPH radical scavenging activity up to 95% and consequently, the lowest IC50 was found for skin of Ghara Shani. According to RP-HPLC experiments, the highest concentration of phenolic compounds was identified as catechin (945 μg/g), epicatechin (482 μg/g), gallic acid (319 μg/g) and resveratrol (29.8 μg/g) in skin of Ghara Shani, quercetin in cane of Ghara Shani (956 μg/g), rutin in skin of Ghara Shira (298 μg/g) and caffeic acid in cane of Ghara Shira (17.4 μg/g). PMID:26776043

  3. ATP-competitive LRRK2 inhibitors interfere with monoclonal antibody binding to the kinase domain of LRRK2 under native conditions. A method to directly monitor the active conformation of LRRK2?

    PubMed

    Gillardon, Frank; Kremmer, Elisabeth; Froehlich, Thomas; Ueffing, Marius; Hengerer, Bastian; Gloeckner, Christian J

    2013-03-30

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions. PMID:23318290

  4. 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. PMID:22251726

  5. 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

  6. Characterization of native Bacillus thuringiensis strains and selection of an isolate active against Spodoptera frugiperda and Peridroma saucia.

    PubMed

    Alvarez, Analía; Virla, Eduardo G; Pera, Licia M; Baigorí, Mario D

    2009-12-01

    Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate--Bt RT--produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity. PMID:19693442

  7. Two cell wall Kunitz trypsin inhibitors in chickpea during seed germination and seedling growth.

    PubMed

    Hernández-Nistal, Josefina; Martín, Ignacio; Jiménez, Teresa; Dopico, Berta; Labrador, Emilia

    2009-03-01

    Two Kunitz trypsin inhibitors TPI-1 and TPI-2, encoded by CaTPI-1 and CaTPI-2, previously identified and characterized, have been detected in chickpea (Cicer arietinum L.) embryonic axes from seeds imbibed up to 48 h. Their gene transcription commenced before germination sensu stricto was completed. The transcript amount of CaTPI-1 remained high until 24 h after imbibition, when the epicotyls started to grow, while CaTPI-2 mRNA, which appeared later, reached a maximum at 48 h. Both the temporal and the spatial distribution of TPI-1 and TPI-2 proteins in the embryonic axes suggest that they perform different functions. The early appearance of TPI-1 in imbibed seeds suggests that it plays a protective role, preventing the premature degradation of the proteins stored in the embryonic axes. Its pattern of distribution suggests that the protein is involved in the regulation of vascular tissue differentiation, protecting the cells from some proteinases involved in programmed cell death. With regard to TPI-2, its later synthesis after imbibition, together with its tissue distribution, indicates that it is mainly active following germination, during elongation of the embryonic axes. PMID:19091584

  8. Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

    PubMed

    Gupta, Munishwar N; Jain, Sulakshana; Roy, Ipsita

    2002-01-01

    Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. PMID:11822903

  9. The synthesis of lysylfluoromethanes and their properties as inhibitors of trypsin, plasmin and cathepsin B.

    PubMed Central

    Angliker, H; Wikstrom, P; Rauber, P; Shaw, E

    1987-01-01

    The synthesis of two lysylfluoromethanes is described by an extension of the synthesis method of Rauber, Angliker, Walker & Shaw [(1986) Biochem. J. 239, 633-640]. Ala-Phe-Lys-CH2F was found to be an active-centre-directed inhibitor of plasmin and trypsin, as is the corresponding chloromethane. However, the rate of covalent-bond formation is about an order of magnitude lower at 25 degrees C for the fluoro derivative. It was, in addition, an extremely effective inactivator of cathepsin B at pH 5.4 and 6.4. The chemical reactivity of fluoromethanes was compared with that of chloromethanes as alkylators of GSH. At pH 7.4 and 37 degrees C, a fluoromethane has 1/500th the reactivity of a chloromethane. A comparison of the rates of reaction of the fluoromethane with cathepsin B and with GSH at pH 6.4 revealed an enhancement of 10(8)-fold for the alkylation of the enzyme, ascribable largely to a proximity effect. PMID:2954536

  10. Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis

    PubMed Central

    DUHAIME, MICHAEL J.; PAGE, KHALIPH O.; VARELA, FAUSTO A.; MURRAY, ANDREW S.; SILVERMAN, MICHAEL E.; ZORATTI, GINA L.; LIST, KARIN

    2016-01-01

    Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. PMID:26297835

  11. Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

    PubMed

    Duhaime, Michael J; Page, Khaliph O; Varela, Fausto A; Murray, Andrew S; Silverman, Michael E; Zoratti, Gina L; List, Karin

    2016-07-01

    Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. PMID:26297835

  12. Auditory–motor interactions for the production of native and non-native speech

    PubMed Central

    Jones, ‘Ōiwi Parker; Seghier, Mohamed L.; Duncan, Keith J. Kawabata; Leff, Alex P.; Green, David W.; Price, Cathy J.

    2013-01-01

    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 fMRI and analysed with dynamic causal modelling (DCM). 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. PMID:23392667

  13. Calculation of protein conformation as an assembly of stable overlapping segments: application to bovine pancreatic trypsin inhibitor.

    PubMed Central

    Simon, I; Glasser, L; Scheraga, H A

    1991-01-01

    Conformations of bovine pancreatic trypsin inhibitor were calculated by assuming that the final structure as well as properly chosen overlapping segments thereof are simultaneously in low-energy (not necessarily the lowest-energy) conformational states. Therefore, the whole chain can be built up from building blocks whose conformations are determined primarily by short-range interactions. Our earlier buildup procedure was modified by taking account of a statistical analysis of known amino acid sequences that indicates that there is nonrandom pairing of amino acid residues in short segments along the chain, and by carrying out energy minimization on only these segments and on the whole chain [without minimizing the energies of intermediate-size segments (20-30 residues long)]. Results of this statistical analysis were used to determine the variable sizes of the overlapping oligopeptide building blocks used in the calculations; these varied from tripeptides to octapeptides, depending on the amino acid sequence. Successive stages of approximations were used to combine the low-energy conformations of these building blocks in order to keep the number of variables in the computations to a manageable size. The calculations led to a limited number of conformations of the protein (only two different groups, with very similar structure within each group), most residues of which were in the same conformational state as in the native structure. PMID:2023916

  14. 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

  15. Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na sup + + K sup + )-ATPase

    SciTech Connect

    Bayer, R. )

    1990-03-06

    The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the {alpha}-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact {alpha}-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na{sup +} + K{sup +})-ATPase were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin. The labeled {alpha}-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold int the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin.

  16. 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.

  17. A label-free fluorescence assay for trypsin based on the electron transfer between oligonucleotide-stabilized Ag nanoclusters and cytochrome c.

    PubMed

    Hong, Mei-Lan; Li, Li-Juan; Han, Hui-Xia; Chu, Xia

    2014-01-01

    A label-free fluorescent assay for the detection of trypsin by using oligonucleotide-templated silver nanoclusters (Ag NCs) and cytochrome c (Cyt c) has been demonstrated. When negatively charged Ag NCs and positively charged Cyt c are mixed, they tend to form a hybrid complex, and then lead the fluorescence of Ag NCs to be quenched significantly due to electron transfer between Ag NCs and the heme cofactor of Cyt c. In the presence of trypsin, it catalyzes the hydrolytic cleavage of Cyt c to small peptide fragments, and releases the heme moiety from the Ag NCs/Cyt c complex; the quenched fluorescence restores therewith. By virtue of this specific response, the fluorescent biosensor has a linear range of from 0.7 to 4 μg mL(-1) and from 9 to 120 μg mL(-1) with a detection limit of 58.7 ng mL(-1). Aside from the easy manufacture aspect, our method also possesses a high signal-to-background ratio (~11), excellent selectivity and good biocompatibility, which makes it a promising bioanalysis for a trypsin activity assay. PMID:25109643

  18. Cloning and sequencing of the blood meal-induced late trypsin gene from the mosquito Aedes aegypti and characterization of the upstream regulatory region.

    PubMed

    Barillas-Mury, C; Wells, M A

    1993-01-01

    A 4.1 kb genomic clone of the late trypsin gene from the mosquito Aedes aegypti was isolated, mapped and subcloned. A 1.6 kb subclone, corresponding to 1.1 kb of upstream regulatory region and 0.5 kb of coding region, was sequenced. The gene has no introns within the coding region. The 5' end of the mature mRNA was mapped using primer extension analysis. A TATA box consensus sequence (TATAAA) was found at position -31 from the 5' end of the mature mRNA. A cluster of five repeat sequences homologous to the yeast GCN4 DNA binding site was found within 200 nucleotides upstream of the cap site. GCN4 is required for derepression mediated control of general amino acid biosynthesis in response to amino acid starvation in yeast. It activates the transcription of at least twenty different genes coding for enzymes involved in amino acid biosynthesis. The presence of this cluster of consensus sequences suggests that a protein similar to GCN4 might regulate expression of the late trypsin gene in the mosquito. Southern blot analysis of genomic DNA indicates that late trypsin is a single copy gene. PMID:9087537

  19. 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. PMID:18381266

  20. 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. PMID:18581136

  1. Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads.

    PubMed

    Malmsten; Xing; Ljunglöf

    1999-12-15

    The immobilization of trypsin on porous glycidyl methacrylate (GMA-GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple "contrast matching" method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media. Copyright 1999 Academic Press. PMID:10607463

  2. 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.

  3. Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk.

    PubMed

    Marchbank, Tania; Weaver, Gillian; Nilsen-Hamilton, Marit; Playford, Raymond J

    2009-04-01

    Colostrum is the first milk produced after birth and is rich in immunoglobulins and bioactive molecules. We examined whether human colostrum and milk contained pancreatic secretory trypsin inhibitor (PSTI), a peptide of potential relevance for mucosal defense and, using in vitro and in vivo models, determined whether its presence influenced gut integrity and repair. Human milk was collected from individuals over various times from parturition and PSTI concentrations determined with the use of immunoassay. Human milk samples were analyzed for proliferation and promigratory activity (wounded monolayers) and antiapoptotic activity (caspase-3 activity) with the use of intestinal HT29 cells with or without neutralizing antibodies to PSTI and epidermal growth factor (EGF). Rats were restrained and given indomethacin to induce gastric injury. Effect of gavage with human breast milk with or without neutralizing antibodies on amount of injury were compared with animals receiving a commercial formula feed. PSTI is secreted into human milk, with colostrum containing a much higher concentration of PSTI than human milk obtained later. Human milk stimulated migration and proliferation about threefold and reduced indomethacin-induced apoptosis by about 70-80%. Sixty-five percent of the migratory effect of human milk could be removed by immunoneutralization of PSTI. PSTI worked synergistically with EGF in mediating these effects. Gastric damage in rats was reduced by about 75% in the presence of human milk and was more efficacious than the formula feed (P<0.001). Protective effects of the milk were reduced by about 60% by PSTI immunoneutralization. We concluded that PSTI is secreted into human milk at concentrations that have probable pathophysiological relevance. PMID:19147803

  4. Building Native Nations through Native Student's Commitment to Their Communities

    ERIC Educational Resources Information Center

    Lee, Tiffany S.

    2009-01-01

    One aspect of building Native nations entails motivating American Indian/Alaska Native youth to become committed to their communities so as to sustain and move forward with the goals of American Indian/Alaska Native nations. This study determined the impact of one Native American Studies department on its Native students' life goals. Through its…

  5. Ins(1,4,5)P3 interacts with PIP2 to regulate activation of TRPC6/C7 channels by diacylglycerol in native vascular myocytes

    PubMed Central

    Ju, Min; Shi, Jian; Saleh, Sohag N; Albert, Anthony P; Large, William A

    2010-01-01

    We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P3 at 10 μm increased 3-fold TRPC6-like activity induced by 10 μm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P3 had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 μm reduced phosphatidylinositol 4,5-bisphosphate (PIP2) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP2 antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP2, a water-soluble PIP2 analogue, inhibited OAG-induced TRPC6/C7 activity with an IC50 of 0.74 μm. Ins(1,4,5)P3 rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP2 in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P3 receptor antagonist heparin. In contrast, Ins(1,4,5)P3 did not overcome diC8-PIP2-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P3/AM (6-Ins(1,4,5)P3), a cell-permeant analogue of Ins(1,4,5)P3, at 10 μm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP2, but not TRPC6 and PIP2. In contrast, 10 μm OAG reduced association between TRPC6 and PIP2, but not between TRPC7 and PIP2. The present work provides the first evidence that Ins(1,4,5)P3 modulates native TRPC channel activity through removal of the

  6. Efficacy of Trypsin in Treating Coral Snake Envenomation in the Porcine Model.

    PubMed

    Parker-Cote, Jennifer L; O'Rourke, Dorcas P; Brewer, Kori L; Lertpiriyapong, Kvin; Punja, Mohan; Bush, Sean P; Miller, Susan N; Meggs, William J

    2015-12-01

    Antivenom is the definitive treatment for venomous snakebites. Alternative treatments warrant investigation because antivenom is sometimes unavailable, expensive, and can have deleterious side effects. This study assesses the efficacy of trypsin to treat coral snake envenomation in an in vivo porcine model. A randomized, blinded study was conducted. Subjects were 13 pigs injected subcutaneously with 1 mL of eastern coral snake venom (10 mg/mL) in the right distal hind limb. After 1 min, subjects were randomized to have the envenomation site injected with either 1 mL of saline or 1 mL of trypsin (100 mg/mL) by a blinded investigator. Clinical endpoint was survival for 72 h or respiratory depression defined as respiratory rate <15 breaths per minute, falling pulse oximetry, or agonal respirations. Fisher's exact t test was used for between group comparisons. Average time to toxicity for the saline control was 263 min (191-305 min). The development of respiratory depression occurred more frequently in control pigs than treated pigs (p = 0.009). Four of the six pigs that received trypsin survived to the end of the 3-day study. No control pigs survived. Two of the trypsin treatment pigs died with times to toxicity of 718 and 971 min. Survival to 12 and 24 h was significantly greater in the trypsin treatment group (p = 0.002, p = 0.009, respectively). Local injection of trypsin, a proteolytic enzyme, at the site of envenomation decreased the toxicity of eastern coral snake venom and increased survival significantly. Further investigation is required before these results can be extended to human snakebites. PMID:25952763

  7. Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG.

    PubMed

    Kotormán, Márta; Simon, Mária L; Borics, Attila; Szabó, Márton Richárd; Szabó, Kitti; Szögi, Titanilla; Fülöp, Lívia

    2015-01-01

    The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol. PMID:26428300

  8. 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.

  9. 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…

  10. AIDS and Native Youth.

    ERIC Educational Resources Information Center

    Luna, G. Cajetan

    Native Americans throughout North America suffer from a greater prevalence of health problems than the population as a whole. One might believe that the problem of AIDS is insignificant for Native youth, but such a belief is inaccurate and shortsighted. As of March 1989, the Centers for Disease Control reported 1,792 cases of childhood and…

  11. Native Speaker Insight

    ERIC Educational Resources Information Center

    Broughton, Geoffrey

    1978-01-01

    Defines the concept of native speaker insight and suggests that, for the purpose of teaching English as a second language, the goal should not be native speaker insight (NSI) but NS Type 1, a reduced, adequate and attainable goal for foreign learners. (CFM)

  12. Listen to the Natives

    ERIC Educational Resources Information Center

    Prensky, Marc

    2006-01-01

    "Digital natives" refer to today's students because they are native speakers of technology, fluent in the digital language of computers, video games, and the Internet. Those who were not born into the digital world are referred to as digital immigrants. Educators, considered digital immigrants, have slid into the 21st century--and into the digital…

  13. 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,…

  14. Following Native Language Acquisition.

    ERIC Educational Resources Information Center

    Neiburg, Michael S.

    Native language acquisition is a natural and non-natural stage-by-stage process. The natural first stage is development of speech and listening skills. In this stage, competency is gained in the home environment. The next, non-natural stage is development of literacy, a cultural skill taught in school. Since oral-aural native language development…

  15. Native SAD is maturing.

    PubMed

    Rose, John P; Wang, Bi-Cheng; Weiss, Manfred S

    2015-07-01

    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

  16. 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

  17. [Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)].

    PubMed

    Kuznetsova, S S; Kolesanova, E F; Talanova, A V; Veselovsky, A V

    2016-05-01

    Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given. PMID:27562989

  18. Chinese Tallow Trees (Triadica sebifera) from the Invasive Range Outperform Those from the Native Range with an Active Soil Community or Phosphorus Fertilization

    PubMed Central

    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/m2), phosphorus (control or 0.5 g/m2), 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, an

  19. 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

  20. Identification and transcription profiling of Trypsin in Aedes taeniorhynchus (Diptera: Culicidae): Developmental regulation, blood feeding, and Permethrin exposure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA of a trypsin gene from Aedes (Ochlerotatus) taeniorhynchus (Weidemann) was cloned and sequenced. The full-length mRNA sequence (874 bp) for trypsin from Ae. taeniorhynchus (AetTryp) was obtained which encodes an open reading frame of 717 bp (i.e., 239 aa). To detect whether AetTryp is devel...

  1. The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

    PubMed

    Gomis-Rüth, F X; Gómez, M; Bode, W; Huber, R; Avilés, F X

    1995-09-15

    The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far. PMID:7556081

  2. Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

    PubMed

    Ferry, N; Jouanin, L; Ceci, L R; Mulligan, E A; Emami, K; Gatehouse, J A; Gatehouse, A M R

    2005-01-01

    Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus. PMID:15643975

  3. 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.

  4. Possible peroxidase active site environment in amyloidogenic proteins: Native monomer or misfolded-oligomer; which one is susceptible to the enzymatic activity, with contribution of heme?

    PubMed

    Khodarahmi, Reza; Ashrafi-Kooshk, Mohammad Reza; Khodarahmi, Sina; Ghadami, Seyyed Abolghasem; Mostafaie, Ali

    2015-09-01

    Amyloid states of many proteins complex with heme and exhibit significant non-specific peroxidase activity, compared to free heme. Neurotransmitter deficiency, generation of neurotoxins, altered activity/metabolism of key enzymes and cellular DNA damage are possible evidences highlighting the importance of the uncontrollable peroxidase activity in Alzheimer's disease (AD)-involved brain cells. Despite extensive experimental work was carried out on this field, discrepancy on chronological precedence of amyloid aggregation and oxidative reactions as well as the mechanism involved in the peroxidase-induced oxidative stress is still not completely understood. In this study, we highlight further that heme cofactor readily complexes with structural intermediates of amyloid aggregates of ovalbumin, lactoglobulin and crystallin and report the ability of "heme-amyloid aggregate/oligomer") to produce peroxidase-like active site. Histidine side chains are also proposed as both distal and proximal residues required for proper function of these peroxidase systems. Taking uncontrollable peroxidase activity of "Aβ-heme" complex into account, it appears that this process, as a new opened dimension in AD pathologic research, provides structural/mechanistic basis for more efficient therapeutic strategies against neurodegenerative diseases. PMID:26123814

  5. [Development of new soybean germplasm with null lipoxygenase and Kunitz trypsin inhibitor genes].

    PubMed

    Han, Fen-Xia; Ding, An-Lin; Sun, Jun-Ming; Li, Gui-Ying

    2005-04-01

    Soybean is one of the most important sources of plant protein for human. Soybean protein is a kind of high-quality protein composed of balanced amino acids, which contains all kinds of amino acids, especially 8 amino acids necessary for human. But it also contains some components that are not good for human and affect food quality, such as lipoxygenase (Lox) and trypsin inhibitor (Ti). Those are important anti-nutritious factors. Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, new soybean germplasms that pyramided multiple genes of high quality, null lipoxygenase and trypsin inhibitor genes (Ix1, Ix2, Ix3 and ti) were developed by means of cross and biochemical marker-assisted selection of progenies for null lipoxygenase and trypsin inhibitor genes using known Lox and Ti markers (protein markers). Female parents were soybean varieties Ludou 4, Zhongpin 661, Yudou 8,91D15, wei8640 popularized in Huanghuaihai Plain. Male parents were varieties introduced from US, trypsin-inhibitor (Ti)-deficient varieties P. I. L83-4387 and near isogenic lines of varieties Century for lipoxygenase (Lox)-deficient genes, Century-2 (Ix2), Century-2.3 (Ix2Ix3) and Century-1.3 (Ix1 Ix3). These new germplasms will promote soybean breeding for improved quality production,and utilization. PMID:16011034

  6. A randomized controlled trial of trypsin to treat brown recluse spider bites in Guinea pigs.

    PubMed

    Cabaniss, Wyman W; Bush, Sean; O'Rourke, Dorcas P; Fletcher, Paul F; Brewer, Kori L; Lertpiriyapong, Kvin; Punja, Mohan; Miller, Susan N; Meggs, William J

    2014-09-01

    Brown recluse spider bites result in necrotic skin lesions for which there is no known antidote. Since venom toxins are proteins, a proteolytic enzyme like trypsin might be effective in reducing toxicity. The aim of this study was to conduct a randomized controlled trial of trypsin to treat brown recluse spider bites in guinea pigs. Subjects were 18 female guinea pigs. Anesthesia for injections was inhaled isoflurane. Analgesia was 0.05 mg/kg of buprenorphine twice a day as needed. Intervention was intradermal injection of 30 μg of brown recluse venom (Spider Pharm, Yarnell, AZ). Immediately after envenomation, subjects were randomized to two groups of nine: trypsin 10 μg in 1 mL normal saline and 1 mL of normal saline. The primary outcome was lesion area over a 10-day time period. Statistical analysis was performed with repeated measures ANOVA. Mean lesion area was smaller but not statistically different in the placebo group. Maximum lesion size occurred at day 4 in both groups, when lesion area was 76.1 ± 108.2 mm(2) in the placebo group and 149.7 ± 127.3 mm(2) in the treatment group. P value was 0.15 for placebo vs. treatment. This study did not establish a role for trypsin as a treatment for brown recluse spider bites in a guinea pig model. PMID:24849803

  7. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other...

  8. 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... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other...

  9. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other...

  10. 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... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other...

  11. 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 storytelling, and decision-making skills. After conducting five train-the-trainer sessions for Native youth, staff were invited by youth participants to implement the full curriculum as a pilot test at one tribal community site in the Pacific Northwest. Implementation was accompanied by surveys and weekly participant observations and was followed by an interactive meeting to assess youth engagement, determine project acceptability, and solicit suggestions for curriculum changes. Results Six youths aged 12 to 15 (average age = 14) participated in the Native Comic Book Project. Youth participants stated that they liked the project and gained knowledge of the harmful effects of commercial tobacco use but wanted better integration of comic book creation, decision making, and Native storytelling themes. Conclusion Previous health-related comic book projects did not recruit youth as active producers of content. This curriculum shows promise as a culturally appropriate intervention to help Native youth adopt healthy decision-making skills and healthy behaviors by creating their own comic books. PMID:22259070

  12. Knots in rings. The circular knotted protein Momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate.

    PubMed

    Cemazar, Masa; Daly, Norelle L; Häggblad, Sara; Lo, Kai Pong; Yulyaningsih, Ernie; Craik, David J

    2006-03-24

    The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys1-Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability. PMID:16547012

  13. Depth and orientational dependencies of MRI T2 and T1ρ sensitivities towards trypsin degradation and Gd-DPTA2− presence in articular cartilage at microscopic resolution

    PubMed Central

    Wang, Nian; Xia, Yang

    2011-01-01

    Depth and orientational dependencies of microscopic MRI T2 and T1ρ sensitivities were studied in native and trypsin-degraded articular cartilage, before and after being soaked in 1 mM Gd-DTPA2− solution. When the cartilage surface was perpendicular to B0, a typical laminar appearance was visible in T2 weighted images but not in T1ρ weighted images, especially when the spin-lock field was high (2 kHz). At the magic angle (55°) orientation, neither T2 nor T1ρ weighted image had a laminar appearance. Trypsin degradation caused a depth and orientational dependent T2 increase (4–64%) and a more uniform T1ρ increase at a sufficiently high spin-lock field (55–81%). The presence of the Gd ions caused both T2 and T1ρ 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 T1ρ measurements (without and with the presence of the Gd ions) was proposed to be a new imaging marker for cartilage degradation. PMID:22244543

  14. Calnexin and calreticulin bind to enzymically active tissue-type plasminogen activator during biosynthesis and are not required for folding to the native conformation.

    PubMed Central

    Allen, S; Bulleid, N J

    1997-01-01

    The roles of the endoplasmic-reticulum lectins calnexin and calreticulin in the folding of tissue-type plasminogen activator (tPA) have been investigated using an in vitro translation system that reconstitutes these processes as they would occur in the intact cell. Using co-immunoprecipitation of newly synthesized tPA with antibodies to calnexin and calreticulin, it was demonstrated that the interaction of tPA with both lectins was dependent upon tPA glycosylation and glucosidase trimming. When tPA was synthesized in the presence of semi-permeabilized cells under conditions preventing complex formation with calnexin and calreticulin, the translation product had a specific plasminogenolytic activity identical with that when synthesized under conditions permitting interactions with both lectins. Furthermore, complexes of tPA bound to calnexin and calreticulin were shown to be enzymically active. These results demonstrate that calnexin and calreticulin can form a stable interaction with correctly folded tPA; however, such interactions are not required for the synthesis of enzymically active tPA. PMID:9359841

  15. 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. PMID:26639991

  16. Native American Tribal Websites.

    ERIC Educational Resources Information Center

    Miller, Eric L.

    1999-01-01

    Lists Web sites maintained by 38 different Native American nations that deal with topics ranging from tribal history, news, arts and crafts, tourism, entertainment, and commerce. Represented nations include Apache, Blackfeet, Creek, Iroquois, Mohegan, and Sioux. (CMK)

  17. Native American Identity

    ERIC Educational Resources Information Center

    Horse, Perry G.

    2005-01-01

    Many issues and elements--including ethnic nomenclature, racial attitudes, and the legal and political status of American Indian nations and Indian people--influence Native American identity. (Contains 3 notes.)

  18. Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

    PubMed

    Tsilikounas, E; Kettner, C A; Bachovchin, W W

    1992-12-29

    We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1463754

  19. In vitro and in vivo cytotoxic activity of native and ricin conjugated monoclonal antibodies to HBs antigen for Alexander primary liver cell carcinoma cells and tumours.

    PubMed Central

    Oladapo, J M; Goodall, A H; de Koning, R; Parmar, J; Brown, D; Thomas, H C

    1984-01-01

    In in vitro and in vivo systems, native or ricin conjugated monoclonal anti-HBs, are capable of inhibiting or slowing the growth of Alexander primary hepatocellular carcinoma cells. Failure of the immune response to this component of the hepatitis B virus may be one permissive factor in the development of some primary liver cell carcinoma in chronic HBV carriers. PMID:6329921

  20. Diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus Opuntia humifusa (Cactaceae) from the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 total of 108 endophytic fungal isolates were obtained and identified by molecular methods into 17 different taxa of the gen...