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Sample records for aspartic proteinases fourier

  1. Secreted Aspartic Proteinase Family of Candida tropicalis

    PubMed Central

    Zaugg, Christophe; Borg-von Zepelin, Margarete; Reichard, Utz; Sanglard, Dominique; Monod, Michel

    2001-01-01

    Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis λEMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection. PMID:11119531

  2. Isolation and characterization of recombinant Drosophila Copia aspartic proteinase.

    PubMed

    Athauda, Senarath B P; Yoshioka, Katsuji; Shiba, Tadayoshi; Takahashi, Kenji

    2006-11-01

    The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects. PMID:16813567

  3. New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

    PubMed

    Volkov, D A; Dergousova, N I; Rumsh, L D

    2004-06-01

    This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry. PMID:15236611

  4. Synthesis and proteinase inhibitory properties of diphenyl phosphonate analogues of aspartic and glutamic acids.

    PubMed

    Hamilton, R; Walker, B; Walker, B J

    1998-07-01

    The synthesis of diphenyl phosphonate analogues of aspartic and glutamic acid, and their inhibitory activity against S. aureus V8 protease and granzyme B, is described. The study has revealed difficulties with protecting group compatibility in the synthesis of these analogues. Two analogues, Acetyl. AspP (OPh)2 and Acetyl.GluP (OPh)2 were found to function as irreversible inactivators of V8 proteinase, yet exhibit no activity against granzyme B. PMID:9873408

  5. An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features.

    PubMed

    ten Have, Arjen; Dekkers, Ester; Kay, John; Phylip, Lowri H; van Kan, Jan A L

    2004-07-01

    Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1-5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen. PMID:15256589

  6. Key features determining the specificity of aspartic proteinase inhibition by the helix-forming IA3 polypeptide.

    PubMed

    Winterburn, Tim J; Wyatt, David M; Phylip, Lowri H; Bur, Daniel; Harrison, Rebecca J; Berry, Colin; Kay, John

    2007-03-01

    The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained. PMID:17145748

  7. Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn.

    PubMed

    Winterburn, Tim J; Wyatt, David M; Phylip, Lowri H; Berry, Colin; Bur, Daniel; Kay, John

    2006-08-01

    In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur. PMID:16895485

  8. N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity.

    PubMed

    Winterburn, Tim J; Phylip, Lowri H; Bur, Daniel; Wyatt, David M; Berry, Colin; Kay, John

    2007-07-01

    Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein. PMID:17608726

  9. A Novel Aspartic Proteinase-Like Gene Expressed in Stratified Epithelia and Squamous Cell Carcinoma of the Skin

    PubMed Central

    Rhiemeier, Verena; Breitenbach, Ute; Richter, Karl Hartmut; Gebhardt, Christoffer; Vogt, Ingeborg; Hartenstein, Bettina; Fürstenberger, Gerhard; Mauch, Cornelia; Hess, Jochen; Angel, Peter

    2006-01-01

    Homeostasis of stratified epithelia, such as the epidermis of the skin, is a sophisticated process that represents a tightly controlled balance between proliferation and differentiation. Alterations of this balance are associated with common human diseases including cancer. Here, we report the cloning of a novel cDNA sequence, from mouse back skin, that is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and codes for a hitherto unknown aspartic proteinase-like protein (Taps). Taps represents a potential AP-1 target gene because TPA-induced expression in epidermal keratinocytes critically depends on c-Fos, and co-treatment with dexamethasone, a potent inhibitor of AP-1-mediated gene regulation, resulted in impaired activation of Taps expression. Taps mRNA and protein are restricted to stratified epithelia in mouse embryos and adult tissues, implicating a crucial role for this aspartic proteinase-like gene in differentiation and homeostasis of multilayered epithelia. During chemically induced carcinogenesis, transient elevation of Taps mRNA and protein levels was detected in benign skin tumors. However, its expression is negatively associated with dedifferentiation and malignant progression in squamous cell carcinomas of the skin. Similar expression was observed in squamous skin tumors of patients, suggesting that detection of Taps levels represents a novel strategy to discriminate the progression state of squamous skin cancers. PMID:16565508

  10. Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

    PubMed

    Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-Ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

    2016-04-01

    Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians. PMID:26711235

  11. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    PubMed

    Torruella, M; Gordon, K; Hohn, T

    1989-10-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed. PMID:2684630

  12. The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

    PubMed Central

    Xie, Sancai; Green, Jonathan; Bixby, James B.; Szafranska, Bozena; DeMartini, James C.; Hecht, Steven; Roberts, R. Michael

    1997-01-01

    The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions. PMID:9371757

  13. Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

    PubMed

    Gogol, Mariusz; Ostrowska, Dominika; Klaga, Kinga; Bochenska, Oliwia; Wolak, Natalia; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej; Rapala-Kozik, Maria

    2016-01-01

    Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense. PMID:26641639

  14. Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy

    NASA Technical Reports Server (NTRS)

    Maeda, A.; Sasaki, J.; Shichida, Y.; Yoshizawa, T.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K.

    1992-01-01

    The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.

  15. Aspartic acid

    MedlinePlus

    ... also called asparaginic acid. Aspartic acid helps every cell in the body work. It plays a role in: Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as ...

  16. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  17. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  18. Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

    PubMed

    Amarant, T; Burkhart, W; LeVine, H; Arocha-Pinango, C L; Parikh, I

    1991-08-30

    The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents. PMID:1911844

  19. Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae).

    PubMed

    Lufrano, Daniela; Faro, Rosário; Castanheira, Pedro; Parisi, Gustavo; Veríssimo, Paula; Vairo-Cavalli, Sandra; Simões, Isaura; Faro, Carlos

    2012-09-01

    Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases. PMID:22727116

  20. Multiple forms of calcium-dependent proteinase in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1986-01-01

    Four calcium-dependent proteinase (CDP) activities in lobster muscles have been resolved by high performance liquid chromatography. These activities differ in molecular weight and net charge. Though optimum activity occurred at high (5 and 10 mM) calcium at pH 6.8, the enzymes differ in activation at lower calcium concentrations. Only one of the CDPs is active at 100 ..mu..M calcium; none are active at 10 ..mu..M and below. Although all four CDPs are inhibited by the cysteine proteinase inhibitors leupeptin, E-64, and iodoacetamide, they show a differential response to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor PMSF. In contrast to CDPs from vertebrate tissues, crustacean muscles contain multiple forms that require calcium at millimolar levels. 17 refs., 6 figs.

  1. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  2. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  3. [Proteinase-proteinase inhibitor complex in rats under oxidative stress caused by administration of cobalt chloride].

    PubMed

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2000-01-01

    Mechanisms of proteinase-inhibitor proteinase system response was estimated following of cobalt chloride injection. The increase proteinase activity, which led to significant decrease of alpha-2-macroglobulin (alpha-2-MG) level was established that indicated to the removal of the proteinase in complex with alpha-2-MG from the organism. Increase of alpha-1-proteinase inhibitor (alpha-1-PI) trypsin-inhibitory activity in the kidneys testify about removal of oxidative alpha-1-PI. PMID:10979565

  4. Characterization of proteinases in trypanosomatids.

    PubMed

    Branquinha, M H; Vermelho, A B; Goldenberg, S; Bonaldo, M C

    1994-02-01

    Proteinases are important factors in the pathogenicity of many parasitic diseases. In this study, the proteolytic activities of 10 trypanosomatids from five different genera (Crithidia, Phytomonas, Endotrypanum, Trypanosoma and Leishmania) were determined by SDS-PAGE containing copolymerized gelatin as substrate. In almost all species we could detect two proteolytic classes, cysteine- and metalloproteinases, based on the inhibition of their activities by E-64 and 1,10-phenanthroline, respectively. In all cases, the metalloproteinase activities did not change over a broad pH range (from 5.5 to 10). E. schaudinni, T. mega, T. dionisii, C. luciliae, C. fasciculata, C. oncopelti and C. guilhermei expressed one or two metalloproteinases of 45-66 kDa, whereas in P. serpens and P. hyssopifolia a double band of this endopeptidase was detected at 94 kDa. In contrast, no metalloproteinase activity was observed in L. tarentolae. The optimal pH for the cysteine-proteinase activities was acidic (about 5.5). In E. schaudinni, T. mega and in Crithidia sp., these proteinases had an apparent molecular weight of 66-94 kDa, while L. tarentolae expressed a broad band from 29 to 45 kDa. In Phytomonas sp., this class of endopeptidase showed a unique feature, in that major cysteine-proteinases were found at 29-66 kDa, but multiple, low-activity bands were detected from 116 to 200 kDa. The most striking characteristic, however, was the very intense cysteine-proteinase activity expressed by T. dionisii (29-66 kDa). We conclude that these differences in the proteolytic profiles could be useful markers to characterize and compare trypanosomatids. PMID:8081271

  5. AN INACTIVE PRECURSOR OF STREPTOCOCCAL PROTEINASE

    PubMed Central

    Elliott, Stuart D.; Dole, Vincent P.

    1947-01-01

    1. Streptococcal proteinase is derived from an inactive precursor found in culture filtrates of proteinase-producing streptococci. 2. The precursor can be converted into the proteinase by low concentrations of trypsin but not by chymotrypsin. 3. In cultures grown in suitable media the conversion of precursor to proteinase is effected autocatalytically. This reaction occurs under reducing conditions and is initiated by active proteinase present in low concentrations with the precursor. 4. The autocatalytic reaction is suppressed or retarded by conditions which decrease the activity of the proteinase, e.g. by growing cultures at 22°C. instead of at 37°C. or by growing them under markedly aerobic conditions. It is also retarded in the presence of casein. PMID:19871616

  6. A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

    PubMed Central

    Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

    1998-01-01

    A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

  7. Structure of equine infectious anemia virus proteinase complexed with an inhibitor.

    PubMed Central

    Gustchina, A.; Kervinen, J.; Powell, D. J.; Zdanov, A.; Kay, J.; Wlodawer, A.

    1996-01-01

    Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well. PMID:8844837

  8. Reactive oxygen species and anti-proteinases.

    PubMed

    Siddiqui, Tooba; Zia, Mohammad Khalid; Ali, Syed Saqib; Rehman, Ahmed Abdur; Ahsan, Haseeb; Khan, Fahim Halim

    2016-01-01

    Reactive oxygen species (ROS) cause damage to macromolecules such as proteins, lipids and DNA and alters their structure and function. When generated outside the cell, ROS can induce damage to anti-proteinases. Anti-proteinases are proteins that are involved in the control and regulation of proteolytic enzymes. The damage caused to anti-proteinase barrier disturbs the proteinase-anti-proteinases balance and uncontrolled proteolysis at the site of injury promotes tissue damage. Studies have shown that ROS damages anti-proteinase shield of the body by inactivating key members such as alpha-2-macroglobulin, alpha-1-antitrypsin. Hypochlorous acid inactivates α-1-antitrypsin by oxidizing a critical reactive methionine residue. Superoxide and hypochlorous acid are physiological inactivators of alpha-2-macroglobulin. The damage to anti-proteinase barrier induced by ROS is a hallmark of diseases such as atherosclerosis, emphysema and rheumatoid arthritis. Thus, understanding the behaviour of ROS-induced damage to anti-proteinases may helps us in development of strategies that could control these inflammatory reactions and diseases. PMID:26699123

  9. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  10. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. PMID:2327970

  11. Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis.

    PubMed

    Lun, H M; Mak, C H; Ko, R C

    2003-05-01

    Inhibitor sensitivity assays using azocaesin and FTC-caesin as substrates showed that the excretory/secretory (E/S) products of the infective-stage larvae of Trichinella spiralis contained serine, metallo-, cysteine and aspartic proteinases. The activity of the metallo-proteinase was zinc ion dependent (within a range of ZnSO(4) concentrations). Gelatin-substrate gel electrophoresis revealed two bands of molecular mass 48 and 58 kDa which were sensitive to the metallo-proteinase inhibitor EDTA. The former peptide was probably a cleavage product of the latter. The authenticity of the 58 kDa metallo-proteinase as an E/S product was confirmed by immunoprecipitation. Using PCR and RACE reactions, a complete nucleotide sequence of the metallo-proteinase gene was obtained. It comprised 2,223 bp with an open reading frame encoding 604 amino acid residues. The 3' untranslated region consisted of 352 bp, including a polyadenylation signal AATAA. A consensus catalytic zinc-binding motif was present. The conserved domains suggest that the cloned metallo-proteinase belongs to the astacin family and occurs as a single copy gene with 11 introns and 10 exons. Cluster analysis showed that the sequence of the metallo-proteinase gene of T. spiralis resembles those of Caenorhabdites elegans and Strongyloides stercoralis. PMID:12743801

  12. [Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

    PubMed

    Dunaevskiĭ, Ia E; Gruban', T N; Beliakova, G A; Belozerskiĭ, M A

    2006-01-01

    The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen). PMID:17205798

  13. Monoclonal antibodies to the two most basic papaya proteinases.

    PubMed

    Goodenough, P W; Kilshaw, P J; McEwan, F; Owen, A J

    1986-08-01

    The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A. PMID:3545314

  14. Rapid kinetic studies and structural determination of a cysteine proteinase mutant imply that residue 158 in caricain has a major effect upon the ability of the active site histidine to protonate a dipyridyl probe.

    PubMed

    Katerelos, N A; Goodenough, P W

    1996-11-26

    Cysteine proteinases are endopeptidases whose catalytic activity depends upon the nucleophilicity of the active site cysteine thiol group. An ion pair forms with an active site histidine. The presence in some cysteine proteinases of an aspartic acid close to the ion pair has been used as evidence of a "catalytic triad" as found in the serine proteinases. In these enzymes, the correct alignment of serine, histidine, and aspartate residues controls catalysis. However, the absence of the homologous aspartate residue in the mammalian cysteine proteinases cathepsins B and H argues against this pivotal role for aspartic acid. Instead, an Asn, physically close to the histidine in cysteine proteinases, has been proposed as a member of the catalytic triad. Protein engineering is being used to investigate these questions. In this study, the Asp158Glu mutant of the plant cysteine proteinase caricain was analyzed by stopped-flow rapid kinetics. The probe that was used was 2,2'-dipyridyl disulfide (2 PDS), and the profile of k versus pH gave results more closely allied to a small molecule active site model than the normal profile with cysteine proteinases. Multiple pKa's identified in the profile are as follows: pK1 = 3.4 (Cys 25), pK2 = 3.6, pK3 = 7.0, and pK4 = 8.6 (His 158). The structure of the enzyme with the bound inhibitor E64 was solved (R factor of 19.3%). Although the distance between the imadazolium and the surrounding charged amino acids is only slightly changed in the mutant, the reduced steady state activity and narrower pH range can be related to changes in the hydrogen-bonding capacity of the imadazolium. PMID:8942638

  15. Molecular-Scale Study of Aspartate Adsorption on Goethite and Competition with Phosphate.

    PubMed

    Yang, Yanli; Wang, Shengrui; Xu, Yisheng; Zheng, Binghui; Liu, Jingyang

    2016-03-15

    Knowledge of the interfacial interactions between aspartate and minerals, especially its competition with phosphate, is critical to understanding the fate and transport of amino acids in the environment. Adsorption reactions play important roles in the mobility, bioavailability, and degradation of aspartate and phosphate. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) measurements and density functional theory (DFT) calculations were used to investigate the interfacial structures and their relative contributions in single-adsorbate and competition systems. Our results suggest three dominant mechanisms for aspartate: bidentate inner-sphere coordination involving both α- and γ-COO(-), outer-sphere complexation via electrostatic attraction and H-bonding between aspartate NH2 and goethite surface hydroxyls. The interfacial aspartate is mainly governed by pH and is less sensitive to changes of ionic strength and aspartate concentration. The phosphate competition significantly reduces the adsorption capacity of aspartate on goethite. Whereas phosphate adsorption is less affected by the presence of aspartate, including the relative contributions of diprotonated monodentate, monoprotonated bidentate, and nonprotonated bidentate structures. The adsorption process facilitates the removal of bioavailable aspartate and phosphate from the soil solution as well as from the sediment pore water and the overlying water. PMID:26870876

  16. [Character of changes in indicators of proteinase and proteinase inhibitor activity in gastroenterological pathology in children].

    PubMed

    Dovgun, O B; Tebloeva, L T; Shumeĭko, N K; Rudenskaia, G N

    1998-01-01

    The aim of this study was to determine trypsin-lake proteinase activity, chymotrypsin-like proteinase activity, trypsin, alpha 1-antitrypsin and alpha 2-macroglobulin levels in blood serum at the children with gastroenterological pathology. These parameters did not chang at the children with functional disorder of stomach and duodenum. The stable balance between proteinases and inhibitors was determined only at the duration of the disease not more 5 years. The absence of normal levels these enzymes after traditional treatment was explain the necessity to continue the therapy at home with control of enzymes' levels. PMID:9703629

  17. [Effect of adrenal stress on activity of proteinase and alpha-1-proteinase inhibitor in rats].

    PubMed

    Samokhina, L M; Kaliman, P A

    1994-01-01

    The effect of adrenal stress on the proteinase and alpha-1-proteinase inhibitor activities in blood serum and cytosols of the rat organs were investigated. The reliable change was marked only in the alpha-1-PI level research of lung tissue cytosol. The proteolysis suppression was revealed in the heart and kidney tissue, while the proteolysis activation was revealed in serum and less in the lung tissue cytosol. Changes in proteinase level in the myocardium and kidney tissue play the primary role in respect to those of the other research liquids under study. PMID:7747353

  18. Insulin Aspart (rDNA Origin) Injection

    MedlinePlus

    ... unless it is used in an external insulin pump. In patients with type 2 diabetes, insulin aspart ... also can be used with an external insulin pump. Before using insulin aspart in a pump system, ...

  19. Inhibition of proteinase K by phosphorylated sugars.

    PubMed

    Orstan, A; Gafni, A

    1991-11-01

    Proteolysis of lactate dehydrogenase, aldolase and the synthetic substrate N-succinylalanylalanylalanyl-p-nitroanilide by proteinase K is inhibited by glucose-6-phosphate and fructose-1,6-biphosphate. Analysis of the kinetic data obtained with the synthetic substrate indicates that the inhibition is a mixed-type and that more than one inhibitor molecule binds to proteinase K. Glucose and fructose are ineffective as inhibitors. In the presence of 0.2-4 mM fructose-1,6-biphosphate, aldolase becomes more susceptible to proteolysis, probably as a result of a conformational change induced by the substrate. PMID:1815500

  20. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of...

  1. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of...

  2. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of...

  3. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of...

  4. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of...

  5. Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. a paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

    PubMed Central

    Olson, Steven T.; Richard, Benjamin; Izaguirre, Gonzalo; Schedin-Weiss, Sophia; Gettins, Peter G. W.

    2010-01-01

    Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors. PMID:20685328

  6. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor.

    PubMed

    Prasad, C V S Siva; Gupta, Saurabh; Gaponenko, Alex; Tiwari, Murlidhar

    2013-08-01

    Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein-protein binding sites were predicted. Protein-protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG-VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops. PMID:23813339

  7. Novel proteinase inhibitor promotes resistance to insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

  8. Aspartate protects Lactobacillus casei against acid stress.

    PubMed

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-05-01

    The aim of this study was to investigate the effect of aspartate on the acid tolerance of L. casei. Acid stress induced the accumulation of intracellular aspartate in L. casei, and the acid-resistant mutant exhibited 32.5 % higher amount of aspartate than that of the parental strain at pH 4.3. Exogenous aspartate improved the growth performance and acid tolerance of Lactobacillus casei during acid stress. When cultivated in the presence of 50 mM aspartate, the biomass of cells increased 65.8 % compared with the control (without aspartate addition). In addition, cells grown at pH 4.3 with aspartate addition were challenged at pH 3.3 for 3 h, and the survival rate increased 42.26-fold. Analysis of the physiological data showed that the aspartate-supplemented cells exhibited higher intracellular pH (pHi), intracellular NH4 (+) content, H(+)-ATPase activity, and intracellular ATP pool. In addition, higher contents of intermediates involved in glycolysis and tricarboxylic acid cycle were observed in cells in the presence of aspartate. The increased contents of many amino acids including aspartate, arginine, leucine, isoleucine, and valine in aspartate-added cells may contribute to the regulation of pHi. Transcriptional analysis showed that the expression of argG and argH increased during acid stress, and the addition of aspartate induced 1.46- and 3.06-fold higher expressions of argG and argH, respectively, compared with the control. Results presented in this manuscript suggested that aspartate may protect L. casei against acid stress, and it may be used as a potential protectant during the production of probiotics. PMID:23292549

  9. Predicting proteinase specificities from free energy calculations.

    PubMed

    Mekonnen, Seble Merid; Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2006-10-01

    The role of the primary binding residue (P1) in complexes between three different subtilases (subtilisin Carlsberg, thermitase and proteinase K) and their canonical protein inhibitor eglin c have been studied by free energy calculations. Based on the crystal structures of eglin c in complex with subtilisin Carlsberg and thermitase, and a homology model of the eglin c-proteinase K complex, a total of 57 mutants have been constructed and docked into their host proteins. The binding free energy was then calculated using molecular dynamics (MD) simulations combined with the linear interaction energy (LIE) method for all complexes differing only in the nature of the amino acid at the P1 position. LIE calculations for 19 different complexes for each subtilase were thus carried out excluding proline. The effects of substitutions at the P1 position on the binding free energies are found to be very large, and positively charged residues (Arg, Lys and His) are particularly deleterious for all three enzymes. The charged variants of the acidic side chains are found to bind more favorably as compared to their protonated states in all three subtilases. Furthermore, hydrophobic amino acids are accommodated most favorably at the S1-site in all three enzymes. Comparison of the three series of binding free energies shows only minor differences in the 19 computed relative binding free energies among these subtilases. This is further reflected in the correlation coefficient between the 23 relative binding free energies obtained, including the possible protonation states of ionizable side chains, but excluding the P1 Pro, for subtilisin Carlsberg versus thermitase (0.95), subtilisin versus proteinase K (0.94) and thermitase versus proteinase K (0.96). PMID:16386933

  10. Role for different cell proteinases in cancer invasion and cytolysis.

    PubMed Central

    Zucker, S.; Beck, G.; DiStefano, J. F.; Lysik, R. M.

    1985-01-01

    The crucial role of non-plasminogen dependent serine proteinases is tissue invasive and cytolytic functions of Walker 256 cancer cells has been documented using a rat urinary bladder invasion and a 125I-labelled fibroblast cytolysis assay. The invasive capacity of these cancer cells was abrogated by non toxic concentrations of the serine proteinase inhibitors, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, but not by metallo or cysteine proteinase inhibitors. Although tumour cell collagenase activity and plasminogen activator were demonstrated, these proteolytic enzymes were not essential in these in vitro assays. These results suggest that different categories of proteinases play specific roles in the complicated process of cancer invasion. PMID:2992566

  11. [Effect of pentoxyphylline on certain indicators of the proteinase-proteinase inhibitor system in rats upon administration of cycloheximide].

    PubMed

    Samokhin, A A; Kaliman, P A; Samokhinka, L M

    2001-01-01

    The pentoxifylline influence on neutral proteinase, alpha-2-macroglobulin, trypsin-alpha-1-proteinase inhibitor and elastaseinhibitory activity under cycloheximide injection has been investigated. Two hours after cycloheximide injection the activity of neutral proteinases increases in rats serum, lungs, heart, liver and kidneys. The preliminary injection of pentoxifylline prevents increase of neutral proteinases activity. Cycloheximide also decreases alpha-2-macroglobulin activity in serum and liver and trypsin-, elastaseinhibitory activity of alpha-1-proteinase inhibitor in all investigated organs. At using pentoxifylline the alpha-2-macroglobulin activity doesn't change in liver and increases in serum in comparison with only cycloheximide and there are no observed any alpha-1 inhibitor proteinase activity changes in rats serum and organs. PMID:12035527

  12. Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1982-01-01

    The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D.L. Mykles and D.M. Skinner, 1981, J. Ultrastruct. Res. 75, 314 to 325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca/sup 2 +/ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of crysteine proteinases, leupeptin, and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

  13. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    SciTech Connect

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  14. Nitrification of Aspartate by Aspergillus flavus

    PubMed Central

    Hatcher, H. J.; Schmidt, E. L.

    1971-01-01

    Heterotrophic conversion of l-aspartic acid to nitrification products by Aspergillus flavus was studied in a replacement incubation system. Numerous amino acids supported nitrification; aspartate and glutamate were about equivalent as the best sources of nitrate. Addition of sodium bicarbonate to the incubation system substantially enhanced nitrate formation for all nitrifiable amino acids except aspartic acid, but the basis for the bicarbonate effect is obscure. The yield of nitrate from l-aspartate was not approached by forms of aspartic acid resulting from substitution on the beta carbon, the amino nitrogen, or the gamma carboxyl group or by aspartate presented as the d-configuration. There was no relationship between nitrate formation and the occurrence of such possible intermediates as nitrite, bound hydroxylamine, ammonia, aspergillic acid, and beta-nitropropionic acid. Uniformly labeled 14C-l-aspartate that was nitrified in replacement incubation led to no accumulation of label in possible nitrification products in the culture filtrate. Label was found in components of the mycelium after acid hydrolysis, with heaviest accumulation in what appeared to be glucosamine and an unidentified compound, possibly acetylglucosamine. Detectable label was redistributed into serine, glycine, and threonine. Images PMID:5549699

  15. Insulin Aspart (rDNA Origin) Injection

    MedlinePlus

    ... a solution (liquid) and a suspension (liquid with particles that will settle on standing) to inject subcutaneously ( ... it is colored, cloudy, thickened, or contains solid particles. If you are using insulin aspart suspension, the ...

  16. Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

    PubMed Central

    Owen, Caroline A.

    2008-01-01

    A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

  17. The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases.

    PubMed Central

    Rauber, P; Angliker, H; Walker, B; Shaw, E

    1986-01-01

    A synthesis of peptidylfluoromethanes is described that utilizes the conversion of phthaloyl amino acids into their fluoromethane derivatives. These can be deblocked and elongated. The inactivation of chymotrypsin by Cbz-Phe-CH2F (benzyloxycarbonylphenylalanylfluoromethane) was found to be considerably slower than that of the analogous chloromethane. The fluoromethane analogue inactivates chymotrypsin with an overall rate constant that is 2% of that observed for the inactivation of the enzyme with the chloromethane. However, the result is the same. The reagent complexes in a substrate-like manner, with Ki = 1.4 X 10(-4) M, and alkylates the active-centre histidine residue. Cbz-Phe-Phe-CH2F and Cbz-Phe-Ala-CH2F were investigated as inactivators of the cysteine proteinase cathepsin B. The difference in reactivity between fluoromethyl ketones and chloromethyl ketones is less pronounced in the case of the cysteine proteinase than for the serine proteinase. Covalent bond formation takes place in this case also, as demonstrated by the use of a radiolabelled reagent. PMID:3827817

  18. Fractionation and purification of the thiol proteinases from papaya latex.

    PubMed

    Dekeyser, P M; De Smedt, S; Demeester, J; Lauwers, A

    1994-06-01

    Three cysteine proteinases, i.e. chymopapain, papaya proteinase IV and proteinase III, were purified to homogeneity from papaya latex using a combination of ion-exchange chromatography and hydrophobic interaction chromatography. During the purification procedure, the thiol-groups of the active center were reversibly blocked as mixed disulfides with 2-thiopyridone. Homogeneity was proved electrophoretically by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and rechromatography on a Mono S 5/5 column at pH 5.0. PMID:7952030

  19. Kinetic analysis of papaya proteinase omega.

    PubMed

    Sumner, I G; Vaughan, A; Eisenthal, R; Pickersgill, R W; Owen, A J; Goodenough, P W

    1993-08-01

    Papaya proteinase omega (pp omega) has been purified from dried latex both by immunoaffinity and traditional methods. Kinetic analysis revealed that (1), the pp omega-catalysed hydrolysis of N-benzoyl-L-arginine p-nitroanilide (BApNA) has a lower specificity (kcat/Km) than the same reaction catalysed by papain; (2), the pp omega-catalysed hydrolysis of a tripeptide substrate having phenylalanine at the second position (S2-site) showed a more similar specificity to that catalysed by papain; (3), the significant difference between the two enzymes is that steady state kinetics with both L-BApNA and a tripeptide enables the identification in pp omega of other ionizations affecting binding. The active sites of papain and pp omega can therefore be distinguished by pH-dependence of kcat/Km. PMID:8393709

  20. [Ammonia release and binding processes and the activity of acid proteinase and glycolysis enzymes in the dynamics of experimental toxic adrenal encephalopathy].

    PubMed

    Kovalenko, V M; Trapezontseva, R A; Vilkov, G A

    1988-01-01

    The content of ammonium, glutamine, glutamate, aspartate and GABA, glutamine synthetase activity, acid proteinase, hexonase, phosphohexoisomerase and dehydrogenase glucose-6-phosphate were studied in dog brain homogenates after individual injections of Bacillus coli endotoxin (10 micrograms/kg) and adrenaline (75 micrograms/kg) into veins and their combined injections into the carotid artery. Isolated injections of endotoxin and adrenaline were shown to cause transient metabolic compensatory changes. Combined injections caused stable progressing brain metabolic disorders. It is suggested that neurochemical changes influence endogenous development of toxic adrenal encephalopathy. PMID:3276357

  1. Isolation and preliminary characterization of the cysteine-proteinases from the latex of Carica candamarcensis Hook.

    PubMed

    Walreavens, V; Jaziri, M; van Beeumen, J; Schnek, A G; Kleinschmidt, T; Looze, Y

    1993-07-01

    The cysteine-proteinase chymopapain from Carica papaya L. is used for chemonucleolysis of damaged human intervertebral spinal discs. The purification of this enzyme is difficult. To overcome these problems, we were looking for a substitute among the cysteine-proteinases of Carica candamarcensis Hook. The latex from unripe fruits was collected in an aqueous solution of methylethanethiolsulfonate to prevent proteolytic activities. The soluble fraction of the lypophilized product provided four enzymatically active peaks (CC-I-CC-IV) during chromatography on CM-Sephadex C-50 in sodium acetate buffer, pH5.0. They could be further purified by rechromatography under similar conditions. The isolated enzymes have been characterized by PAGE, analysis of the Fourier transform infrared spectra, preliminary studies of their specificities as well as a comparison of the N-terminal amino-acid sequences up to position 43. CC-III proved to be glycosylated. CC-I and CC-III from Carica candamarcensis Hook are suggested to correspond to papain and chymopapain from Carica papaya L., respectively. PMID:8216902

  2. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  3. A low molecular weight proteinase inhibitor produced by T lymphocytes.

    PubMed Central

    Ganea, D; Teodorescu, M; Dray, S

    1986-01-01

    A low molecular weight (MW) proteinase inhibitor, between 6500 and 21,500 MW, appeared in the supernatant of rabbit spleen cells cultured at high density for 24 hr. The inhibitor inhibited the enzymatic activity of trypsin for both a high MW natural substrate, fibrinogen, and for a low MW artificial substrate, Chromozym TRY. The low MW proteinase inhibitor is protein in nature and is different, in terms of specificity for enzymes, MW and sensitivity to different physical or chemical treatments, from aprotinin, a low MW proteinase inhibitor (6500 MW) of bovine origin, and from the soybean trypsin inhibitor, a relatively high MW proteinase inhibitor (21,500 MW). The inhibitor was found in the supernatant of purified T cells but not B cells, and its production was increased in the presence of an optimal concentration of Con A. The possibility that this proteinase inhibitor has a role in the regulation of trypsin-like proteinases involved to the immune response remains to be investigated. Images Figure 4 PMID:2417942

  4. The induction of proteinases in corn and soybean by anoxia

    SciTech Connect

    VanToai, T.; Hwang, Shihying )

    1989-04-01

    This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with {sup 3}H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia.

  5. [Effect of quercetin on some indicators of the proteinase-proteinase inhibitor system in rats upon administration of cobalt chloride to them].

    PubMed

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2001-01-01

    The results of quercetin effect on some changes of proteinase--proteinase inhibitor system parameters in rats under cobalt chloride injection are shown. It was established that preliminary quercetin administration prevened neutral proteinase activation and alpha-2-macroglobulin activity decreasing after 2 h of cobalt chloride influence. PMID:12199071

  6. An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

    PubMed

    Visal-Shah, S; Vrain, T C; Yelle, T C; Nguyen-Quoc, B; Michaud, D

    2001-08-01

    A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology. PMID:11545387

  7. Fourier Series Operating Package

    NASA Technical Reports Server (NTRS)

    Charnow, Milton L.

    1961-01-01

    This report presents a computer program for multiplying, adding, differentiating, integrating, "barring" and scalarly multiplying "literal" Fourier series as such, and for extracting the coefficients of specified terms.

  8. Proteinases in the joint: clinical relevance of proteinases in joint destruction

    PubMed Central

    Rengel, Yvonne; Ospelt, Caroline; Gay, Steffen

    2007-01-01

    Proteinases are involved in essential steps in cartilage and bone homeostasis. Consequently, efforts have been made to establish their potential role in the pathology of rheumatic conditions such as rheumatoid arthritis, osteoarthritis and spondyloarthritis. Matrix metalloproteinases (MMPs) are sensitive markers of disease severity and response to treatment, and therefore they have potential in the assessment of rheumatic diseases. Despite disappointing early results with synthetic inhibitors of MMPs, there is still much scope for developing effective and safe MMPs inhibitors, and consequently to deliver new options to inhibit joint destruction. PMID:18001502

  9. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    PubMed Central

    2011-01-01

    Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent

  10. Developing novel anthelmintics from plant cysteine proteinases

    PubMed Central

    Behnke, Jerzy M; Buttle, David J; Stepek, Gillian; Lowe, Ann; Duce, Ian R

    2008-01-01

    Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock. PMID:18761736

  11. Jean Baptiste Joseph Fourier

    NASA Astrophysics Data System (ADS)

    Sterken, C.

    2003-03-01

    This paper gives a short account of some key elements in the life of Jean Baptiste Joseph Fourier (1768-1830), specifically his relation to Napoleon Bonaparte. The mathematical approach to Fourier series and the original scepticism by French mathematicians are briefly illustrated.

  12. Fourier Series Optimization Opportunity

    ERIC Educational Resources Information Center

    Winkel, Brian

    2008-01-01

    This note discusses the introduction of Fourier series as an immediate application of optimization of a function of more than one variable. Specifically, it is shown how the study of Fourier series can be motivated to enrich a multivariable calculus class. This is done through discovery learning and use of technology wherein students build the…

  13. The primary structure and characterization of carbohydrate chains of the extracellular glycoprotein proteinase inhibitor from latex of Carica papaya.

    PubMed

    Odani, S; Yokokawa, Y; Takeda, H; Abe, S; Odani, S

    1996-10-01

    A secretory proteinase inhibitor was isolated from the latex of green fruits of papaya (Carica papaya). The protein exhibited stoichiometric inhibition of bovine trypsin and alpha-chymotrypsin by the same site or overlapping binding sites. The complete covalent structure consisting of 184 amino acids and two disulfide bonds was determined by protein analysis. During the structural analysis, a procedure was established to separate very hydrophilic peptides by reverse-phase HPLC. The result revealed that the latex protein belongs to an extensively diverse plant protein family that includes inhibitors of serine, cysteine and aspartic proteases, a taste-modifying protein, wound responsive proteins, storage proteins, amylase inhibitors and even an oxidoreductase. In this superfamily, the latex proteinase inhibitor is most similar to the curious protein, miraculin, which makes sour food taste sweet. Two carbohydrate chains, each probably composed of (mannose)5, (xylose)1, (fucose)0-2, and (N-acetylglucosamine)2 residues, were attached to asparagine 84 and 90. Mass-spectrometric and compositional analysis suggested that they may represent a new class of plant xylose-containing carbohydrate chains with five mannose residues. PMID:8898891

  14. 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

  15. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog.

    PubMed

    Zhao, Y; Botella, M A; Subramanian, L; Niu, X; Nielsen, S S; Bressan, R A; Hasegawa, P M

    1996-08-01

    Diverse functions for three soybean (Glycine max L. Merr.) cysteine proteinase inhibitors (CysPIs) are inferred from unique characteristics of differential regulation of gene expression and inhibitory activities against specific Cys proteinases. Based on northern blot analyses, we found that the expression in leaves of one soybean CysPI gene (L1) was constitutive and the other two (N2 and R1) were induced by wounding or methyl jasmonate treatment. Induction of N2 and R1 transcript levels in leaves occurred coincidentally with increased papain inhibitory activity. Analyses of kinetic data from bacterial recombinant CysPI proteins indicated that soybean CysPIs are noncompetitive inhibitors of papain. The inhibition constants against papain of the CysPIs encoded by the wound and methyl jasmonate-inducible genes (57 and 21 nM for N2 and R1, respectively) were 500 to 1000 times lower than the inhibition constant of L1 (19,000 nM). N2 and R1 had substantially greater inhibitory activities than L1 against gut cysteine proteinases of the third-instar larvae of western corn rootworm and Colorado potato beetle. Cysteine proteinases were the predominant digestive proteolytic enzymes in the guts of these insects at this developmental stage. N2 and R1 were more inhibitory than the epoxide trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane (E-64) against western corn rootworm gut proteinases (50% inhibition concentration = 50, 200, and 7000 nM for N2, R1, and E-64, respectively). However, N2 and R1 were less effective than E-64 against the gut proteinases of Colorado potato beetle. These results indicate that the wound-inducible soybean CysPIs, N2 and R1, function in host plant defense against insect predation, and that substantial variation in CysPI activity against insect digestive proteinases exists among plant CysPI proteins. PMID:8756506

  16. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog.

    PubMed Central

    Zhao, Y; Botella, M A; Subramanian, L; Niu, X; Nielsen, S S; Bressan, R A; Hasegawa, P M

    1996-01-01

    Diverse functions for three soybean (Glycine max L. Merr.) cysteine proteinase inhibitors (CysPIs) are inferred from unique characteristics of differential regulation of gene expression and inhibitory activities against specific Cys proteinases. Based on northern blot analyses, we found that the expression in leaves of one soybean CysPI gene (L1) was constitutive and the other two (N2 and R1) were induced by wounding or methyl jasmonate treatment. Induction of N2 and R1 transcript levels in leaves occurred coincidentally with increased papain inhibitory activity. Analyses of kinetic data from bacterial recombinant CysPI proteins indicated that soybean CysPIs are noncompetitive inhibitors of papain. The inhibition constants against papain of the CysPIs encoded by the wound and methyl jasmonate-inducible genes (57 and 21 nM for N2 and R1, respectively) were 500 to 1000 times lower than the inhibition constant of L1 (19,000 nM). N2 and R1 had substantially greater inhibitory activities than L1 against gut cysteine proteinases of the third-instar larvae of western corn rootworm and Colorado potato beetle. Cysteine proteinases were the predominant digestive proteolytic enzymes in the guts of these insects at this developmental stage. N2 and R1 were more inhibitory than the epoxide trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane (E-64) against western corn rootworm gut proteinases (50% inhibition concentration = 50, 200, and 7000 nM for N2, R1, and E-64, respectively). However, N2 and R1 were less effective than E-64 against the gut proteinases of Colorado potato beetle. These results indicate that the wound-inducible soybean CysPIs, N2 and R1, function in host plant defense against insect predation, and that substantial variation in CysPI activity against insect digestive proteinases exists among plant CysPI proteins. PMID:8756506

  17. The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast.

    PubMed Central

    Angliker, H; Wikstrom, P; Shaw, E; Brenner, C; Fuller, R S

    1993-01-01

    Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'control' proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase. PMID:8328974

  18. Imaging Fourier transform spectrometer

    SciTech Connect

    Bennett, C.L.

    1993-09-13

    This invention is comprised of an imaging Fourier transform spectrometer having a Fourier transform infrared spectrometer providing a series of images to a focal plane array camera. The focal plane array camera is clocked to a multiple of zero crossing occurrences as caused by a moving mirror of the Fourier transform infrared spectrometer and as detected by a laser detector such that the frame capture rate of the focal plane array camera corresponds to a multiple of the zero crossing rate of the Fourier transform infrared spectrometer. The images are transmitted to a computer for processing such that representations of the images as viewed in the light of an arbitrary spectral ``fingerprint`` pattern can be displayed on a monitor or otherwise stored and manipulated by the computer.

  19. Fourier Transform Mass Spectrometry

    PubMed Central

    Scigelova, Michaela; Hornshaw, Martin; Giannakopulos, Anastassios; Makarov, Alexander

    2011-01-01

    This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook. PMID:21742802

  20. Effects of leupeptin on proteinase and germination of castor beans

    SciTech Connect

    Alpi, A.; Beevers, H.

    1981-10-01

    Leupeptin, tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloid-storage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.

  1. Generalized fiber Fourier optics.

    PubMed

    Cincotti, Gabriella

    2011-06-15

    A twofold generalization of the optical schemes that perform the discrete Fourier transform (DFT) is given: new passive planar architectures are presented where the 2 × 2 3 dB couplers are replaced by M × M hybrids, reducing the number of required connections and phase shifters. Furthermore, the planar implementation of the discrete fractional Fourier transform (DFrFT) is also described, with a waveguide grating router (WGR) configuration and a properly modified slab coupler. PMID:21686007

  2. A Short Biography of Joseph Fourier and Historical Development of Fourier Series and Fourier Transforms

    ERIC Educational Resources Information Center

    Debnath, Lokenath

    2012-01-01

    This article deals with a brief biographical sketch of Joseph Fourier, his first celebrated work on analytical theory of heat, his first great discovery of Fourier series and Fourier transforms. Included is a historical development of Fourier series and Fourier transforms with their properties, importance and applications. Special emphasis is made…

  3. Antidepressant activity of aspartic acid derivatives.

    PubMed

    Petrov, V I; Sergeev, V S; Onishchenko, N V; Piotrovskii, L B

    2001-04-01

    Antidepressant activity of N-phenyl(benzyl)amino derivatives of aspartic acid was studied on various experimental models of depression. IEM-1770 (30 mg/kg) and IEM-1944 (20 mg/kg) exhibited antidepressant activity after single injection in the forced swimming and tail suspension tests. Antidepressant effect of 14-day administration of these compounds and reference drugs maprotiline (10 mg/kg) and citalopram (10 mg/kg) was confirmed on the model of learned helplessness. PMID:11550022

  4. Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

    NASA Astrophysics Data System (ADS)

    Kudo, Norio; Ataka, Mitsuo; Sasaki, Hiroshi; Muramatsu, Tomonari; Katsura, Tatsuo; Tanokura, Masaru

    1996-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

  5. The thiol proteinases from the latex of Carica papaya L. II. The primary structure of proteinase omega.

    PubMed

    Dubois, T; Kleinschmidt, T; Schnek, A G; Looze, Y; Braunitzer, G

    1988-08-01

    The complete primary structure of the proteinase omega isolated from the latex of the Carica papaya fruits is given. The polypeptide chain contains 216 amino-acid residues, the alignment of which was deduced from sequence analyses of the native enzyme, the tryptic, chymotryptic, peptic and thermolysinolytic peptides and facilitated due to the considerable degree of homology with papain and actinidin. The location of the three disulfide bridges could be established with the help of peptic and thermolysinolytic fragments. Proteinase omega shares 148 identical amino-acid residues (68.5%) with papain and 108 ones (50%) with actinidin, including the three disulfide bridges and the free cysteine residue required for activity, as well as most of the other amino-acid residues involved in the catalytic mechanism and two thirds of the glycine residues which are of structural significance. The homology with other cysteine proteinases of different origin is discussed. PMID:3063283

  6. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

    PubMed Central

    Grakoui, A; McCourt, D W; Wychowski, C; Feinstone, S M; Rice, C M

    1993-01-01

    Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. Images PMID:8386278

  7. Cloning, expression and characterization of the proteinase from human herpesvirus 6.

    PubMed Central

    Tigue, N J; Matharu, P J; Roberts, N A; Mills, J S; Kay, J; Jupp, R

    1996-01-01

    After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site. PMID:8648756

  8. Serine proteinases from barley malt may degrade beta-amylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley seed proteinases are critically important to seed germination and malting in that they generate amino acids from seed N reserves, supporting embryo growth during germination and yeast fermentation during brewing. However, relatively little is known regarding the endogenous protein substrate ...

  9. FUSARIUM SPECIES SYNTHESIZE ALKALINE PROTEINASES IN INFESTED BARLEY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, `scab') is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently involved in plant-microbe interactions, because they degrade the storag...

  10. Identification and characterization of cysteine proteinases of Trypanosoma evansi.

    PubMed

    Yadav, S C; Kumar, R; Kumar, S; Tatu, U; Singh, R K; Gupta, A K

    2011-09-01

    Trypanosoma evansi is a causative agent of 'surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands

  11. Purification and characterization of a novel cysteine proteinase (periodontain) from Porphyromonas gingivalis. Evidence for a role in the inactivation of human alpha1-proteinase inhibitor.

    PubMed

    Nelson, D; Potempa, J; Kordula, T; Travis, J

    1999-04-30

    Periodontal disease is characterized by inflammation of the periodontium manifested by recruitment of neutrophils, which can degranulate, releasing powerful proteinases responsible for destruction of connective tissues, and eventual loss of tooth attachment. Although the presence of host proteinase inhibitors (serpins) should minimize tissue damage by endogenous proteinases, this is not seen clinically, and it has been speculated that proteolytic inactivation of serpins may contribute to progression of the disease. A major pathogen associated with periodontal disease is the Gram-negative anaerobe Porphyromonas gingivalis, and in this report, we describe a novel proteinase that has been isolated from culture supernatants of this organism that is capable of inactivating the human serpin, alpha1-proteinase inhibitor, the primary endogenous regulator of human neutrophil elastase. This new enzyme, referred to as periodontain, belongs to the cysteine proteinase family based on inhibition studies and exists as a 75-kDa heterodimer. Furthermore, periodontain shares significant homology to streptopain, a proteinase from Streptococcus pyogenes, and prtT, a putative proteinase from P. gingivalis. Clearly, the presence of this enzyme, which rapidly inactivates alpha1-proteinase inhibitor, could result in elevated levels of human neutrophil elastase clinically detected in periodontal disease and should be considered as a potential virulence factor for P. gingivalis. PMID:10212191

  12. Fourier plane imaging microscopy

    SciTech Connect

    Dominguez, Daniel Peralta, Luis Grave de; Alharbi, Nouf; Alhusain, Mdhaoui; Bernussi, Ayrton A.

    2014-09-14

    We show how the image of an unresolved photonic crystal can be reconstructed using a single Fourier plane (FP) image obtained with a second camera that was added to a traditional compound microscope. We discuss how Fourier plane imaging microscopy is an application of a remarkable property of the obtained FP images: they contain more information about the photonic crystals than the images recorded by the camera commonly placed at the real plane of the microscope. We argue that the experimental results support the hypothesis that surface waves, contributing to enhanced resolution abilities, were optically excited in the studied photonic crystals.

  13. Introduction to Fourier Optics

    ERIC Educational Resources Information Center

    Huggins, Elisha

    2007-01-01

    Much like a physical prism, which displays the frequency components of a light wave, Fourier analysis can be thought of as a mathematical prism that can tell us what harmonics or frequency components are contained in a recording of a sound wave. We wrote the MacScope II program so that the user could not only see a plot of the harmonic amplitudes…

  14. Fourier Transform Mass Spectrometry.

    ERIC Educational Resources Information Center

    Gross, Michael L.; Rempel, Don L.

    1984-01-01

    Discusses the nature of Fourier transform mass spectrometry and its unique combination of high mass resolution, high upper mass limit, and multichannel advantage. Examines its operation, capabilities and limitations, applications (ion storage, ion manipulation, ion chemistry), and future applications and developments. (JN)

  15. Fourier Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Understanding the global atmospheric changes is difficult with today's current technology. However, with high resolution and nearly continuous observations from a satellite, it's possible to transform our understanding of the atmosphere. To enable the next generation of atmospheric science, a new class of orbiting atmospheric sensors is being developed. The foundation of this advanced concept is the Fourier Transform Spectrometer, or FTS.

  16. The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

    PubMed Central

    Stempniak, M; Hostomska, Z; Nodes, B R; Hostomsky, Z

    1997-01-01

    NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related. PMID:9060645

  17. MOLECULAR CLONING OF TRYPSIN-LIKE CDNAS AND COMPARISON OF PROTEINASE ACTIVITIES IN THE SALIVARY GLANDS AND GUT OF THE TARNISHED PLANT BUG LYGUS LINEOLARIS (HEMIPTERA: MIRIDAE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseina...

  18. Independent task Fourier filters

    NASA Astrophysics Data System (ADS)

    Caulfield, H. John

    2001-11-01

    Since the early 1960s, a major part of optical computing systems has been Fourier pattern recognition, which takes advantage of high speed filter changes to enable powerful nonlinear discrimination in `real time.' Because filter has a task quite independent of the tasks of the other filters, they can be applied and evaluated in parallel or, in a simple approach I describe, in sequence very rapidly. Thus I use the name ITFF (independent task Fourier filter). These filters can also break very complex discrimination tasks into easily handled parts, so the wonderful space invariance properties of Fourier filtering need not be sacrificed to achieve high discrimination and good generalizability even for ultracomplex discrimination problems. The training procedure proceeds sequentially, as the task for a given filter is defined a posteriori by declaring it to be the discrimination of particular members of set A from all members of set B with sufficient margin. That is, we set the threshold to achieve the desired margin and note the A members discriminated by that threshold. Discriminating those A members from all members of B becomes the task of that filter. Those A members are then removed from the set A, so no other filter will be asked to perform that already accomplished task.

  19. Ozone inactivation of human alpha 1-proteinase inhibitor

    SciTech Connect

    Johnson, D.A.

    1980-06-01

    Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tryosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes with serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.

  20. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    PubMed Central

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

  1. Structural characterization of the papaya cysteine proteinases at low pH.

    PubMed

    Huet, Joëlle; Looze, Yvan; Bartik, Kristin; Raussens, Vincent; Wintjens, René; Boussard, Paule

    2006-03-10

    Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration. PMID:16434027

  2. Neutrophil-derived Oxidants and Proteinases as Immunomodulatory Mediators in Inflammation

    PubMed Central

    Witko-Sarsat, V.

    1994-01-01

    Neutrophils generate potent microbicidal molecules via the oxygen-dependent pathway, leading to the generation of reactive oxygen intermediates (ROI), and via the non-oxygen dependent pathway, consisting in the release of serine proteinases and metalloproteinases stored in granules. Over the past years, the concept has emerged that both ROI and proteinases can be viewed as mediators able to modulate neutrophil responses as well as the whole inflammatory process. This is well illustrated by the oxidative regulation of proteinase activity showing that oxidants and proteinases acts is concert to optimize the microbicidal activity and to damage host tissues. ROI and proteinases can modify the activity of several proteins involved in the control of inflammatory process. Among them, tumour necrosis factor-α and interleukin-8, are elective targets for such a modulation. Moreover, ROI and proteinases are also able to modulate the adhesion process of neutrophils to endothelial cells, which is a critical step in the inflammatory process. PMID:18472951

  3. Submicromolar Phosphinic Inhibitors of E. coli Aspartate Transcarbamoylase

    PubMed Central

    Coudray, Laëtitia; Kantrowitz, Evan R.; Montchamp, Jean-Luc

    2009-01-01

    The design, syntheses, and enzymatic activity of two submicromolar competitive inhibitors of aspartate transcarbamoylase (ATCase) are described. The phosphinate inhibitors are analogs of N-phosphonacetyl-L-aspartate (PALA) but have a reduced charge at the phosphorus moiety. The mechanistic implications are discussed in terms of a possible cyclic transition-state during enzymatic catalysis. PMID:19097895

  4. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  5. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  6. Astronomical Fourier spectrometer.

    PubMed

    Connes, P; Michel, G

    1975-09-01

    A high resolution near ir Fourier spectrometer with the same general design as previously described laboratory instruments has been built for astronomical observations at a coudé focus. Present spectral range is 0.8-3.5 microm with PbS and Ge detectors and maximum path difference 1 m. The servo system can accommodate various recording modes: stepping or continuous scan, path difference modulation, sky chopping. A real time computer is incorporated into the system, which has been set up at the Hale 500-cm telescope on Mount Palomar. Samples of the results are given. PMID:20154966

  7. Fourier Domain Sensing

    NASA Technical Reports Server (NTRS)

    Feldkhun, Daniel (Inventor); Wagner, Kelvin H. (Inventor)

    2013-01-01

    Methods and systems are disclosed of sensing an object. A first radiation is spatially modulated to generate a structured second radiation. The object is illuminated with the structured second radiation such that the object produces a third radiation in response. Apart from any spatially dependent delay, a time variation of the third radiation is spatially independent. With a single-element detector, a portion of the third radiation is detected from locations on the object simultaneously. At least one characteristic of a sinusoidal spatial Fourier-transform component of the object is estimated from a time-varying signal from the detected portion of the third radiation.

  8. [Occurrence and function of a proteinase inhibitor in the hemolymph of insects].

    PubMed

    Hanschke, R; Hanschke, M

    1975-01-01

    The presence of proteinase inhibitor has been proved in the hemolymph of a number of insect species from seven different insect orders. The amount of proteinase inhibitor in the hemolymph significantly increases after injection of inactivated bacteria into the hemocoelom of Galleria mellonella-larvae. Moreover the larvae show an increased resistance against normally lethal concentrations of trypsin, chymotrypsin, pronase P and extracellular proteinase produced by Pseudomonas aeruginosa. It is discussed that the proteinase inhibitor is one of the factors acting in the antibacterial defense system in the hemolymph of Galleria mellonella-larvae. PMID:811027

  9. Fourier plane image amplifier

    DOEpatents

    Hackel, L.A.; Hermann, M.R.; Dane, C.B.; Tiszauer, D.H.

    1995-12-12

    A solid state laser is frequency tripled to 0.3 {micro}m. A small portion of the laser is split off and generates a Stokes seed in a low power oscillator. The low power output passes through a mask with the appropriate hole pattern. Meanwhile, the bulk of the laser output is focused into a larger stimulated Brillouin scattering (SBS) amplifier. The low power beam is directed through the same cell in the opposite direction. The majority of the amplification takes place at the focus which is the fourier transform plane of the mask image. The small holes occupy large area at the focus and thus are preferentially amplified. The amplified output is now imaged onto the multichip module where the holes are drilled. Because of the fourier plane amplifier, only about 1/10th the power of a competitive system is needed. This concept allows less expensive masks to be used in the process and requires much less laser power. 1 fig.

  10. Fourier plane image amplifier

    DOEpatents

    Hackel, Lloyd A.; Hermann, Mark R.; Dane, C. Brent; Tiszauer, Detlev H.

    1995-01-01

    A solid state laser is frequency tripled to 0.3 .mu.m. A small portion of the laser is split off and generates a Stokes seed in a low power oscillator. The low power output passes through a mask with the appropriate hole pattern. Meanwhile, the bulk of the laser output is focused into a larger stimulated Brillouin scattering (SBS) amplifier. The low power beam is directed through the same cell in the opposite direction. The majority of the amplification takes place at the focus which is the fourier transform plane of the mask image. The small holes occupy large area at the focus and thus are preferentially amplified. The amplified output is now imaged onto the multichip module where the holes are drilled. Because of the fourier plane amplifier, only .about.1/10th the power of a competitive system is needed. This concept allows less expensive masks to be used in the process and requires much less laser power.

  11. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. PMID:25687923

  12. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    SciTech Connect

    Wu, Lan.

    1989-01-01

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward ({sup 3}H)-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics.

  13. Imaging Fourier Transform Spectrometer

    SciTech Connect

    Bennett, C.L.; Carter, M.R.; Fields, D.J.; Hernandez, J.

    1993-04-14

    The operating principles of an Imaging Fourier Transform Spectrometer (IFTS) are discussed. The advantages and disadvantages of such instruments with respect to alternative imaging spectrometers are discussed. The primary advantages of the IFTS are the capacity to acquire more than an order of magnitude more spectral channels than alternative systems with more than an order of magnitude greater etendue than for alternative systems. The primary disadvantage of IFTS, or FTS in general, is the sensitivity to temporal fluctuations, either random or periodic. Data from the IRIFTS (ir IFTS) prototype instrument, sensitive in the infrared, are presented having a spectral sensitivity of 0.01 absorbance units, a spectral resolution of 6 cm{sup {minus}1} over the range 0 to 7899 cm{sup {minus}1}, and a spatial resolution of 2.5 mr.

  14. Astronomical Fourier spectropolarimetry

    NASA Technical Reports Server (NTRS)

    Forbes, F. F.; Fymat, A. L.

    1974-01-01

    Spectra of the Stokes polarization parameters of Venus (resolution 0.5 per cm) are presented. They were obtained at the Cassegrain focus of the 154-cm telescope of the National Mexican Observatory, Baja California, Mexico, July 12 and 13, 1972, with the Fourier Interferometer Polarimeter (FIP). A preliminary, limited analysis of four spectral features and of the CO2 rotational band structures at 6080 and 6200 per cm has demonstrated that spectral polarization is indeed present. These experimental results, confirmed by two series of observations, provide substantiation for this theoretically predicted phenomenon. They also tend to show that the FIP represents a novel astronomical tool for variable spectral resolution studies of both the intensity and the state of polarization of astronomical light sources.

  15. Fourier Transform Spectrometer System

    NASA Technical Reports Server (NTRS)

    Campbell, Joel F. (Inventor)

    2014-01-01

    A Fourier transform spectrometer (FTS) data acquisition system includes an FTS spectrometer that receives a spectral signal and a laser signal. The system further includes a wideband detector, which is in communication with the FTS spectrometer and receives the spectral signal and laser signal from the FTS spectrometer. The wideband detector produces a composite signal comprising the laser signal and the spectral signal. The system further comprises a converter in communication with the wideband detector to receive and digitize the composite signal. The system further includes a signal processing unit that receives the composite signal from the converter. The signal processing unit further filters the laser signal and the spectral signal from the composite signal and demodulates the laser signal, to produce velocity corrected spectral data.

  16. Rainbow Fourier Transform

    NASA Technical Reports Server (NTRS)

    Alexandrov, Mikhail D.; Cairns, Brian; Mishchenko, Michael I.

    2012-01-01

    We present a novel technique for remote sensing of cloud droplet size distributions. Polarized reflectances in the scattering angle range between 135deg and 165deg exhibit a sharply defined rainbow structure, the shape of which is determined mostly by single scattering properties of cloud particles, and therefore, can be modeled using the Mie theory. Fitting the observed rainbow with such a model (computed for a parameterized family of particle size distributions) has been used for cloud droplet size retrievals. We discovered that the relationship between the rainbow structures and the corresponding particle size distributions is deeper than it had been commonly understood. In fact, the Mie theory-derived polarized reflectance as a function of reduced scattering angle (in the rainbow angular range) and the (monodisperse) particle radius appears to be a proxy to a kernel of an integral transform (similar to the sine Fourier transform on the positive semi-axis). This approach, called the rainbow Fourier transform (RFT), allows us to accurately retrieve the shape of the droplet size distribution by the application of the corresponding inverse transform to the observed polarized rainbow. While the basis functions of the proxy-transform are not exactly orthogonal in the finite angular range, this procedure needs to be complemented by a simple regression technique, which removes the retrieval artifacts. This non-parametric approach does not require any a priori knowledge of the droplet size distribution functional shape and is computationally fast (no look-up tables, no fitting, computations are the same as for the forward modeling).

  17. Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi.

    PubMed

    Porter, P; Susarla, S C; Polikepahad, S; Qian, Y; Hampton, J; Kiss, A; Vaidya, S; Sur, S; Ongeri, V; Yang, T; Delclos, G L; Abramson, S; Kheradmand, F; Corry, D B

    2009-11-01

    Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi. PMID:19710638

  18. COMPARATIVE ANALYSIS OF PROTEINASE ACTIVITIES OF BACILLUS THURINGIENSIS-RESISTANT AND -SUSCEPTIBLE OSTRINIA NUBILALIS (LEPIDOPTERA: CRAMBIDAE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteinase activities were compared in soluble and membrane fractions of gut tissues of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis larvae. The soluble trypsin-like proteinase activity of the resistant strain was reduced 56%, significantly lower than that of the susceptibl...

  19. [Activity of Ca(2+)-dependent neutral proteinases in rat organs under cobalt and mercury chloride injection].

    PubMed

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2003-01-01

    The activity of Ca(2+)-dependent neutral proteinases in rats under cobalt and mercury chloride injection was investigated. The calpains activity increase in the lungs, heart, liver and kidneys was revealed after 2 h cobalt chloride action. The mercury chloride gives a reliable increase of calcium-dependent neutral proteinases only in the kidneys. PMID:14574747

  20. ANALYSIS OF MIDGUT PROTEINASES FROM BACILLUS THURINGIENSIS-SUSCEPTIBLE AND -RESISTANT HELIOTHIS VIRESCENS (LEPIDOPTERA: NOCTUIDAE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) H. virescens strains were compared. The overall pH of gut extracts from YDK and CXC were...

  1. Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease

    PubMed Central

    Owen, Caroline A

    2008-01-01

    Since the early 1960s, a compelling body of evidence has accumulated to show that proteinases play critical roles in airspace enlargement in chronic obstructive pulmonary disease (COPD). However, until recently the causative enzymes and their exact roles in pathologic processes in COPD have not been clear. Recent studies of gene-targeted mice in murine models of COPD have confirmed roles for proteinases not only in airspace enlargement, but also in airway pathologies in COPD. These studies have also shed light on the specific proteinases involved in COPD pathogenesis, and the mechanisms by which these proteinases injure the lung. They have also identified important interactions between different classes of proteinases, and between proteinases and other molecules that amplify lung inflammation and injury. This review will discuss the biology of proteinases and the mechanisms by which they contribute to the pathogenesis of COPD. In addition, I will discuss the potential of proteinase inhibitors and anti-inflammatory drugs as new treatment strategies for COPD patients. PMID:18686734

  2. Quadrature formulas for Fourier coefficients

    NASA Astrophysics Data System (ADS)

    Bojanov, Borislav; Petrova, Guergana

    2009-09-01

    We consider quadrature formulas of high degree of precision for the computation of the Fourier coefficients in expansions of functions with respect to a system of orthogonal polynomials. In particular, we show the uniqueness of a multiple node formula for the Fourier-Tchebycheff coefficients given by Micchelli and Sharma and construct new Gaussian formulas for the Fourier coefficients of a function, based on the values of the function and its derivatives.

  3. Aspartate Aminotransferase in Alfalfa Root Nodules 1

    PubMed Central

    Farnham, Mark W.; Griffith, Stephen M.; Miller, Susan S.; Vance, Carroll P.

    1990-01-01

    Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N2-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N2-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667896

  4. Ultrasonic Transducers for Fourier Analysis.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1995-01-01

    Describes an experiment that uses the ultrasonic transducer for demonstrating the Fourier components of waveshapes such as the square and triangular waves produced by laboratory function generators. (JRH)

  5. An Emerging Role of Degrading Proteinases in Hypertension and the Metabolic Syndrome: Autodigestion and Receptor Cleavage

    PubMed Central

    2011-01-01

    One of the major challenges for hypertension research is to identify the mechanisms that cause the comorbidities encountered in many hypertensive patients, as seen in the metabolic syndrome. An emerging body of evidence suggests that human and experimental hypertensives may exhibit uncontrolled activity of proteinases, including the family of matrix metalloproteinases, recognized for their ability to restructure the extracellular matrix proteins and to play a role in hypertrophy. We propose a new hypothesis that provides a molecular framework for the comorbidities of hypertension, diabetes, capillary rarefaction, immune suppression, and other cell and organ dysfunctions due to early and uncontrolled extracellular receptor cleavage by active proteinases. The proteinase and signaling activity in hypertensives requires further detailed analysis of the proteinase expression, the mechanisms causing proenzyme activation, and identification of the proteinase substrate. This work may open the opportunity for reassessment of old interventions and development of new interventions to manage hypertension and its comorbidities. PMID:22081429

  6. The thiol proteinases from the latex of Carica papaya L. I. Fractionation, purification and preliminary characterization.

    PubMed

    Dubois, T; Jacquet, A; Schnek, A G; Looze, Y

    1988-08-01

    Three thiol proteinases, namely papain, chymopapain and proteinase omega were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as S-sulphenylthiosulfate derivative or after blocking with p-chloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase omega. The active sites of chymopapain and proteinase omega could not be distinguished from that of papain neither by the analysis of the pH dependence of kcat/Km nor by the examination of the pH dependence of the fluorescence emission spectra. PMID:3214554

  7. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

    PubMed Central

    Dougherty, W G; Semler, B L

    1993-01-01

    Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

  8. Fourier Ellipsometry -- An Ellipsometric Approach to Fourier Scatterometry

    NASA Astrophysics Data System (ADS)

    Petrik, P.; Kumar, N.; Fried, M.; Fodor, B.; Juhasz, G.; Pereira, S. F.; Burger, S.; Urbach, H. P.

    2015-01-01

    An extension of Fourier scatterometry is presented, aiming at increasing the sensitivity by measuring the phase difference between the reflections polarized parallel and perpendicular to the plane of incidence. The ellipsometric approach requires no additional hardware elements compared with conventional Fourier scatterometry. Furthermore, incoherent illumination is also sufficient, which enables spectroscopy using standard low-cost light sources.

  9. Crystallographic studies of aspartate racemase from Lactobacillus sakei NBRC 15893.

    PubMed

    Fujii, Tomomi; Yamauchi, Takae; Ishiyama, Makoto; Gogami, Yoshitaka; Oikawa, Tadao; Hata, Yasuo

    2015-08-01

    Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3121, with unit-cell parameters a = b = 104.68, c = 97.29 Å, and diffracted to 2.6 Å resolution. Structure determination is under way. PMID:26249691

  10. Non-enzymic beta-decarboxylation of aspartic acid.

    NASA Technical Reports Server (NTRS)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  11. Activation of Proteinase 3 Contributes to Nonalcoholic Fatty Liver Disease and Insulin Resistance

    PubMed Central

    Toonen, Erik JM; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine TN; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo AB

    2016-01-01

    Activation of inflammatory pathways is known to accompany development of obesity-induced nonalcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive proinflammatory mediators interleukin (IL)-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In this study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human α-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin-resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3-deficient mice showed strongly reduced levels of lipids in the liver after being fed a high-fat diet. Moreover, these mice were resistant to high–fat–diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1–/– mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with α-1 antitrypsin during the last 10 d of a 16-wk high-fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential. PMID:27261776

  12. AN ELECTROPHORETIC STUDY OF A STREPTOCOCCAL PROTEINASE AND ITS PRECURSOR

    PubMed Central

    Shedlovsky, Theodore; Elliott, S. D.

    1951-01-01

    An electrophoretic study of crystalline preparations of a streptococcal proteinase and its precursor established their isoelectric points at pH values of 8.42 and 7.35 respectively (ionic strength 0.10). Preparations of the proteinase appeared to be electrophoretically homogeneous over a pH range of 5 to 8.5. Precursor preparations contained a relatively low concentration of the active enzyme visible as a separate peak in electrophoretic patterns of sufficiently concentrated solutions. Autocatalytic conversion of precursor to active enzyme was complete and resulted in a corresponding change in the electrophoretic pattern. Treatment of precursor preparations with trypsin produced incomplete conversion to the active enzyme and resulted in the formation of a modified precursor protein. This differed from the parent substance in electrophoretic mobility and in susceptibility to trypsin, but resembled it in immunological specificity and, as previously shown, in susceptibility to conversion to active enzyme by autocatalysis. Serological reactions of precursor and active enzyme components withdrawn from the cell after electrophoresis are described. It appears that the precursor protein may have two antigenic groups, one specific, the other shared by the active enzyme which behaves as a single antigen. PMID:14888818

  13. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    SciTech Connect

    Odem, R.R.; Willand, J.L.; Polakoski, K.L. )

    1990-02-01

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

  14. Reconstruction in Fourier space

    NASA Astrophysics Data System (ADS)

    Burden, A.; Percival, W. J.; Howlett, C.

    2015-10-01

    We present a fast iterative fast Fourier transform (FFT) based reconstruction algorithm that allows for non-parallel redshift-space distortions (RSDs). We test our algorithm on both N-body dark matter simulations and mock distributions of galaxies designed to replicate galaxy survey conditions. We compare solenoidal and irrotational components of the redshift distortion and show that an approximation of this distortion leads to a better estimate of the real-space potential (and therefore faster convergence) than ignoring the RSD when estimating the displacement field. Our iterative reconstruction scheme converges in two iterations for the mock samples corresponding to Baryon Oscillation Spectroscopic Survey CMASS Data Release 11 when we start with an approximation of the RSD. The scheme takes six iterations when the initial estimate, measured from the redshift-space overdensity, has no RSD correction. Slower convergence would be expected for surveys covering a larger angle on the sky. We show that this FFT based method provides a better estimate of the real-space displacement field than a configuration space method that uses finite difference routines to compute the potential for the same grid resolution. Finally, we show that a lognormal transform of the overdensity, used as a proxy for the linear overdensity, is beneficial in estimating the full displacement field from a dense sample of tracers. However, the lognormal transform of the overdensity does not perform well when estimating the displacements from sparser simulations with a more realistic galaxy density.

  15. Reflective Fourier ptychography

    NASA Astrophysics Data System (ADS)

    Pacheco, Shaun; Zheng, Guoan; Liang, Rongguang

    2016-02-01

    The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective's NA divided by the wavelength (2NAobj/λ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection.

  16. Reflective Fourier ptychography.

    PubMed

    Pacheco, Shaun; Zheng, Guoan; Liang, Rongguang

    2016-02-01

    The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective's NA divided by the wavelength (2NAobj/λ ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection. PMID:26891601

  17. Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases.

    PubMed Central

    Saklatvala, J; Wood, G C; White, D D

    1976-01-01

    1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme. Images PLATE 2 PLATE 3 PLATE 4 PLATE 5 PLATE 6 PLATE 1 PMID:9069

  18. Explicit Fourier wavefield operators

    NASA Astrophysics Data System (ADS)

    Ferguson, R. J.; Margrave, G. F.

    2006-04-01

    Explicit wavefield extrapolators are based on direct analytic mathematical formulae that express the output as an extrapolation operator acting on the input, while implicit methods usually require the calculation of the numerical inverse of a matrix to obtain the output. Typically, explicit methods are faster than implicit methods, and they often give more insight into the physics of the wave propagation, but they often suffer from instability. Four different explicit extrapolators based on Fourier theory are presented and analysed. They are: PS (ordinary phase shift), GPSPI (generalized phase shift plus interpolation), NSPS (non-stationary phase shift) and SNPS (symmetric non-stationary phase shift). A formal proof is given that NSPS in a direction orthogonal to the velocity gradient is the mathematical adjoint process to GPSPI in the opposite direction. This motivates the construction of SNPS that combines NSPS and GPSPI in a symmetric fashion. This symmetry (under interchange of input and output lateral coordinates) is required by reciprocity arguments. PS and SNPS are symmetric while NSPS and GPSPI are not. A numerical stability study using SVD (singular value decomposition) shows that all of these extrapolators can become unstable for strong lateral velocity gradients. Unstable operators allow amplitudes to grow non-physically in a recursion. Stability is enhanced by introducing a small (~3 per cent) imaginary component to the velocities. This causes a numerical attenuation that tends to stabilize the operators but does not address the cause of the instability. For the velocity model studied (a very challenging case) GPSPI and NSPS have exactly the same instability while SNPS is always more stable. Instability manifests in a complicated way as a function of extrapolation step size, frequency, velocity gradient, and strength of numerical attenuation. The SNPS operator can be stabilized over a wide range of conditions with considerably less attenuation than is

  19. [Aspartate aminotransferase--key enzyme in the human systemic metabolism].

    PubMed

    Otto-Ślusarczyk, Dagmara; Graboń, Wojciech; Mielczarek-Puta, Magdalena

    2016-01-01

    Aspartate aminotransferase is an organ-nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms--cytoplasmic (AST1) and mitochondrial (AST2), that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys - 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp) in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs. PMID:27117097

  20. In situ properties of Helicobacter pylori aspartate carbamoyltransferase.

    PubMed

    Burns, B P; Mendz, G L; Hazell, S L

    1997-11-01

    The kinetic and regulatory properties of aspartate carbamoyltransferase (ACTase) of the human pathogen Helicobacter pylori were studied in situ in cell-free extracts. The presence of enzyme activity was established by identifying the end product as carbamoylaspartate using nuclear magnetic resonance spectroscopy. Activity was measured in all strains studied, including recent clinical isolates. Substrate saturation curves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and aspartate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent Km were 0.6 and 11.6 mm for carbamoyl phosphate and aspartate, respectively. Optimal pH and temperature were determined as 8.0 and 45 degrees C. Activity was observed with the l- but not the d-isomer of aspartate. Succinate and maleate inhibited enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogues acetyl phosphate and phosphonoacetic acid inhibited activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribose 5-phosphate at 10 mm concentration showed 20 and 35% inhibition of enzyme activity, respectively. N-Phosphonoacetyl-l-aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 microM concentration. Inhibition by PALA was competitive with carbamoyl phosphate (Ki = 0.245 microM) and noncompetitive with aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interesting characteristics distinct from this class. PMID:9344472

  1. Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase.

    PubMed Central

    Anjuère, F; Monsigny, M; Lelièvre, Y; Mayer, R

    1993-01-01

    Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared. The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative. The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively. This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis. The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant. The proteinase activity is assessed by measuring the fluorescence of the supernatant. This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range. The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors. PMID:8489513

  2. Limited proteolysis by macrophage elastase inactivities human. cap alpha. /sub 1/-proteinase inhibitor

    SciTech Connect

    Banda, M.J.; Clark, E.J.; Werb, Z.

    1980-12-01

    Ever since the initial description of ..cap alpha../sub 1/-proteinase inhibitor (..cap alpha../sub 1/PI), the role of this plasma glycoprotein and its allelic polymorphism in disease and in healthy physiology has been the subject of much investigation, ..cap alpha../sub 1/PI inactivates a number of serine proteinases, including granulocyte elastase, and thus affords protection from the connective tissue degradation mediated by this class of proteinases. Because an imbalance in the ratio between ..cap alpha../sub 1/PI and proteinase may contribute to the development of destructive lung diseases, proteinases have been implicated in the pathogenesis of pulmonary emphysema. Both macrophages and polymorphonuclear leukocytes have been implicated in disruption of the ..cap alpha../sub 1/PI-proteinase balance. In this report, a new mechanism for alteration of the ..cap alpha../sub 1/PI-proteinase balance is demonstrated. It was found that the purified form of macrophage elastase catalytically degrades and inactivates ..cap alpha../sub 1/PI so that it no longer inhibits the elastinolytic activity of granulocyte elastase.

  3. α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease

    PubMed Central

    Sinden, Nicola J.; Baker, Michael J.; Smith, David J.; Kreft, Jan-Ulrich; Dafforn, Timothy R.

    2014-01-01

    The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor α-1-antitrypsin result from point mutations in its gene. In addition, α-2-macroglobulin competes with α-1-antitrypsin for proteinases, and the α-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of α-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either α-1-antitrypsin or α-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 μm into the tissue before becoming sequestered. We have shown that proteinases sequestered to α-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with α-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all α-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to α-2-macroglobulin where it retains proteolytic activity. PMID:25416382

  4. Fourier phase in Fourier-domain optical coherence tomography

    PubMed Central

    Uttam, Shikhar; Liu, Yang

    2015-01-01

    Phase of an electromagnetic wave propagating through a sample-of-interest is well understood in the context of quantitative phase imaging in transmission-mode microscopy. In the past decade, Fourier-domain optical coherence tomography has been used to extend quantitative phase imaging to the reflection-mode. Unlike transmission-mode electromagnetic phase, however, the origin and characteristics of reflection-mode Fourier phase are poorly understood, especially in samples with a slowly varying refractive index. In this paper, the general theory of Fourier phase from first principles is presented, and it is shown that Fourier phase is a joint estimate of subresolution offset and mean spatial frequency of the coherence-gated sample refractive index. It is also shown that both spectral-domain phase microscopy and depth-resolved spatial-domain low-coherence quantitative phase microscopy are special cases of this general theory. Analytical expressions are provided for both, and simulations are presented to explain and support the theoretical results. These results are further used to show how Fourier phase allows the estimation of an axial mean spatial frequency profile of the sample, along with depth-resolved characterization of localized optical density change and sample heterogeneity. Finally, a Fourier phase-based explanation of Doppler optical coherence tomography is also provided. PMID:26831383

  5. Triacontanol negatively modulates the jasmonic acid-stimulated proteinase inhibitors in tomato (Lycopersicon esculentum).

    PubMed

    Ramanarayan, Krishnamurthy; Swamy, Gangadharamurthy Sivakumar

    2004-04-01

    Triacontanol (TRIA), a long chain aliphatic alcohol (C30H61OH) reverses the effect of jasmonic acid (JA) in inducing proteinase inhibitors (PIs) in tomato leaves. Porcine pancreas trypsin and Spodoptera litura gut proteinases were inhibited in the presence of leaf proteins treated with JA, and TRIA partially reverses this effect. Spodoptera litura larvae fed with tomato leaves treated with JA were reduced in body weight and TRIA is able to partially reverse this JA-induced effect. These results reflect the partial reversal effect of TRIA in down regulating the JA-induced production of proteinase inhibitors. PMID:15128037

  6. Ozone effects on inhibitors of human neutrophil proteinases

    SciTech Connect

    Smith, C.E.; Stack, M.S.; Johnson, D.A.

    1987-02-15

    The effects of ozone on human alpha 1-proteinase inhibitor (A-1-PI), alpha 1-antichymotrypsin (A-1-Achy), bronchial leukocyte proteinase inhibitor (BLPI), and Eglin C were studied using in vitro exposures in phosphate-buffered solutions. Following ozone exposure, inhibitory activities against human neutrophil elastase (HNE) and/or cathepsin G (Cat G) were measured. Exposure of A-1-PI to 50 mol O3/mol protein resulted in a complete loss of HNE inhibitory activity, whereas A-1-Achy lost only 50% of its Cat G inhibitory activity and remained half active even after exposure to 250 mol of O3. At 40 mol O3/mol protein, BLPI lost 79% of its activity against HNE and 87% of its Cat G inhibitory activity. Eglin C, a leech-derived inhibitor, lost 81% of its HNE inhibitory activity and 92% of its ability to inhibit Cat G when exposed to 40 mol O3/mol. Amino acid analyses of ozone-exposed inhibitors showed destruction of Trp, Met, Tyr, and His with as little as 10 mol O3/mol protein, and higher levels of O3 resulted in more extensive oxidation of susceptible residues. The variable ozone susceptibility of the different amino acid residues in the four proteins indicated that oxidation was a function of protein structure, as well as the inherent susceptibility of particular amino acids. Exposure of A-1-PI and BLPI in the presence of the antioxidants, Trolox C (water soluble vitamin E) and ascorbic acid (vitamin C), showed that antioxidant vitamins may protect proteins from oxidative inactivation by ozone. Methionine-specific modification of BLPI reduced its HNE and Cat G inhibitory activities. Two moles of N-chlorosuccinimide per mole of BLPI methionine caused an 80% reduction in activity against Cat G, but only a 40% reduction in HNE inhibitory activity.

  7. 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

  8. Successive Use of Non-Host Plant Proteinase Inhibitors Required for Effective Inhibition of Helicoverpa armigera Gut Proteinases and Larval Growth1

    PubMed Central

    Harsulkar, Abhay M.; Giri, Ashok P.; Patankar, Aparna G.; Gupta, Vidya S.; Sainani, Mohini N.; Ranjekar, Prabhakar K.; Deshpande, Vasanti V.

    1999-01-01

    We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth of Helicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibitor-insensitive proteinases. Electrophoretic analysis of PI-induced HGP showed differential regulation of proteinase isoforms. Interestingly, HGP activity induced in response to dietary potato PI-II was inhibited by winged bean PIs. The optimized combination of potato PI-II and winged bean PIs identified in the present study and their proposed successive use has potential in developing H. armigera-resistant transgenic plants. PMID:10517841

  9. Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.

    PubMed

    Rapala-Kozik, M; Potempa, J; Nelson, D; Kozik, A; Travis, J

    1999-10-01

    Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase. PMID:10595584

  10. Fourier Analysis of Musical Intervals

    NASA Astrophysics Data System (ADS)

    LoPresto, Michael C.

    2008-11-01

    Use of a microphone attached to a computer to capture musical sounds and software to display their waveforms and harmonic spectra has become somewhat commonplace. A recent article in The Physics Teacher aptly demonstrated the use of MacScope2 in just such a manner as a way to teach Fourier analysis.3 A logical continuation of this project is to use MacScope not just to analyze the Fourier composition of musical tones but also musical intervals.

  11. A systematic and mechanistic evaluation of aspartic acid as filler for directly compressed tablets containing trimethoprim and trimethoprim aspartate.

    PubMed

    ElShaer, Amr; Hanson, Peter; Mohammed, Afzal R

    2013-04-01

    The generally accepted paradigm of 'inert' and 'mono functional' excipient in dosage form has been recently challenged with the development of individual excipients capable of exhibiting multiple functions (e.g. binder-disintegrants, surfactant which affect P-gp function). The proposed study has been designed within the realm of multifunctionality and is the first and novel investigation towards evaluation of aspartic acid as a filler and disintegration enhancing agent for the delivery of biopharmaceutical class IV model drug trimethoprim. The study investigated powder characteristics using angle of repose, laser diffractometry and scanning electron microscopy (SEM). The prepared tablets were characterised using Heckel analysis, disintegration time and tensile strength measurements. Although Heckel analysis revealed that both TMP and TMP aspartate salt have high elasticity, the salt form produced a stronger compact which was attributed to the formation of agglomerates. Aspartic acid was found to have high plasticity, but its incorporation into the formulations was found to have a negative impact on the compaction properties of TMP and its salt. Surface morphology investigations showed that mechanical interlocking plays a vital role in binding TMP crystals together during compaction, while the small particle size of TMP aspartate agglomerates was found to have significant impact on the tensile strength of the tablets. The study concluded that aspartic acid can be employed as filler and disintegrant and that compactability within tablets was independent of the surface charge of the excipients. PMID:23207325

  12. Synthesis and In Vitro Evaluation of Aspartate Transcarbamoylase Inhibitors

    PubMed Central

    Coudray, Laëtitia; Pennebaker, Anne F.; Montchamp, Jean-Luc

    2009-01-01

    The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-L-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs. PMID:19828320

  13. Isolation of two plant proteinases in latex from Carica candamarcensis acting as mitogens for mammalian cells.

    PubMed

    Gomes, Marco Túlio R; Mello, Vanessa J; Rodrigues, Kelly C; Bemquerer, Marcelo P; Lopes, Miriam T P; Faça, Vitor M; Salas, Carlos E

    2005-03-01

    In a prior study we showed evidence that latex from Carica candamarcensis contains a protein fraction that stimulates mammalian cell proliferation. In this report we describe the isolation of two proteinases responsible for this effect. Both proteinases (P1, P2) display a relative mass of 23 kDa and following chromatographic purification stimulate proliferation of fibroblastic and epithelial cells. P2 added to L929 fibroblasts at 2.5 nM enhances proliferation by 60 %. We further demonstrate that its cellular effect is linked to an increase in activity of Erk2, a component of the MAP kinase pathway. To our knowledge, this is the first known plant proteinase to exert a proliferative effect in mammalian cells. This novel mitogenic property attributed to a purified cysteine proteinase may explain some of the therapeutic actions attributed to these enzymes. PMID:15770545

  14. Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum.

    PubMed

    Yang, Yurong; Qin, Weiwen; Wei, Hua; Ying, Jianxi; Zhen, Jing

    2011-11-01

    Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum. PMID:21925175

  15. Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.

    PubMed Central

    North, M J; Scott, K I; Lockwood, B C

    1988-01-01

    Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present

  16. Gel Electrophoretic Profiles of Proteinases in Dark-Germinated Flax Seeds 1

    PubMed Central

    Jameel, Shahid; Reddy, V. Manoranjan; Rhodes, W. Gale; McFadden, Bruce A.

    1984-01-01

    The proteinases present in dark-germinated flax seeds (Linum usitatissimum) were studied as a function of germination at 25°C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45°C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedlings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved to be very reproducible and afforded detection and good quantification of various proteinase zones. An ethylenediaminetetraacetate-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). Ethylenediaminetetraacetate also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose-dependent manner. The addition of leupeptin, α-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate, or 1,10-phenanthroline prior to, during, or after exchange of Triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings. Images Fig. 2 Fig. 6 Fig. 7 PMID:16663914

  17. THE CRYSTALLIZATION AND SEROLOGICAL DIFFERENTIATION OF A STREPTOCOCCAL PROTEINASE AND ITS PRECURSOR

    PubMed Central

    Elliott, S. D.

    1950-01-01

    Grown in dialysate broth at a pH between 5.5 and 6.5, some strains of group A streptococci elaborate the precursor of a proteolytic enzyme. Within this range of hydrogen concentration the precursor is also produced when the streptococci are suspended in a peptone dialysate containing glucose and incubated at 37°C. The precursor does not appear to be produced at a neutral or alkaline reaction. Methods are described whereby the precursor and proteinase have been isolated in crystalline form. The precursor crystallizes from half-saturated ammonium sulfate at pH 8.0 and a temperature of 22°C. or higher; the proteinase crystallizes from 0.15 saturated ammonium sulfate at pH 8.0 but does so most readily at refrigerator temperature. The degree of purification achieved by these procedures is discussed. The activity of purified preparations of the precursor and of proteinase has been tested against α-benzoyl-l-arginineamide and, with this as a substrate, the conversion of precursor to proteinase by autocatalysis or by trypsin has been confirmed. Immunological experiments are described, the results of which provide evidence of the distinct antigenic specificity of the precursor and proteinase; the conversion of precursor to proteinase has been followed by means of serological tests. PMID:15436931

  18. Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides.

    PubMed Central

    Markaryan, A; Lee, J D; Sirakova, T D; Kolattukudy, P E

    1996-01-01

    The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion. PMID:8636020

  19. Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

    PubMed Central

    Shahbal, Samaha; Hemme, Denis; Renault, Pierre

    1993-01-01

    The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca2+, Mg2+, and Mn2+ were activators, while Zn2+ and Cu2+ were inhibitors. β-Casein was hydrolyzed more rapidly than αs1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related. Images PMID:16348841

  20. Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme.

    PubMed Central

    Schlick, Petra; Kronovetr, Jakub; Hampoelz, Bernhard; Skern, Tim

    2002-01-01

    The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis. PMID:11964149

  1. A short biography of Joseph Fourier and historical development of Fourier series and Fourier transforms

    NASA Astrophysics Data System (ADS)

    Debnath, Lokenath

    2012-07-01

    The profound study of nature is the most fertile source of mathematical discoveries. Not only does this study, by offering a definite goal to research, have the advantage of excluding vague questions and futile calculations, but it is also a sure means of moulding analysis itself, and discerning those elements in it which it is still essential to know and which science ought to conserve. These fundamental elements are those which recur in all natural phenomena. Joseph Fourier pure mathematics enables us to discover the concepts and laws connecting them, which gives us the key to the understanding of the phenomena of nature. Albert Einstein This article deals with a brief biographical sketch of Joseph Fourier, his first celebrated work on analytical theory of heat, his first great discovery of Fourier series and Fourier transforms. Included is a historical development of Fourier series and Fourier transforms with their properties, importance and applications. Special emphasis is made to his splendid research contributions to mathematical physics, pure and applied mathematics and his unprecedented public service accomplishments in the history of France. This is followed by historical comments about the significant and major impact of Fourier analysis on mathematical physics, probability and mathematical statistics, mathematical economics and many areas of pure and applied mathematics including geometry, harmonic analysis, signal analysis, wave propagation and wavelet analysis. Special attention is also given to the Fourier integral formula, Brownian motion and stochastic processes and many examples of applications including isoparametric inequality, everywhere continuous but nowhere differentiable functions, Heisenberg uncertainty principle, Dirichlets' theorem on primes in

  2. Challenges in pKa Predictions for Proteins: The case of Asp213 in Human Proteinase 3

    NASA Astrophysics Data System (ADS)

    Hajjar, Eric; Dejaegere, Annick; Reuter, Nathalie

    2009-09-01

    Knowledge of the protonation states of the ionizable residues in an enzyme is a prerequisite to an accurate description of its structure and mechanism. In practice, the use of the inappropriate protonation state for an amino acid in a molecular modeling computation (e.g., molecular dynamics simulation) is likely to lead to unrealistic results. Although methods using solvers of the linearized Poisson-Boltzmann equation have proven to yield accurate pKa predictions, they bear a number of limitations. They are quite demanding in terms of computational power and are sensitive to representation of the charges and their position (force field and protein conformation). Moreover they depend on the choice of a dielectric constant for the protein interior. In this manuscript, we describe the difficulties met when trying to predict the protonation state of a buried amino acid, located in a protein for which very little biochemical data is available. Such a case is highly representative of the challenges faced in theoretical biology studies. Proteinase 3 (PR3) is an enzyme involved in proteolytic events associated with inflammation. It is a potential target in the development of new anti-inflammatory therapeutic strategies. We report the results of pKa predictions of the aspartic acid 213 of PR3 with a FDPB solver. We probed the influence of the choice of the dielectric constant for the protein interior ɛp and the benefits of conformational sampling by molecular dynamics (MD) on the pKa prediction of this carboxylate group. Using only the FDPB calculations, we could not conclude on the protonation state of Asp213. MD simulations confronted to knowledge of the ligand-binding and reaction mechanism led us to decide on a protonated form of this aspartic acid. We also demonstrate that the use of the wrong protonation state leads to an unreliable structural model for PR3. pKa prediction with a fast empirical method yielded a pKa of 8.4 for Asp213, which is in agreement with our

  3. Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2000-01-01

    The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress. PMID:11113364

  4. Radiochemical microassay for aspartate aminotransferase activity in the nervous system

    SciTech Connect

    Garrison, D.; Beattie, J.; Namboodiri, M.A.

    1988-07-01

    A radiochemical procedure for measuring aspartate aminotransferase activity in the nervous system is described. The method is based on the exchange of tritium atoms at positions 2 and 3 of L-2,3-(/sup 3/H)aspartate with water when this amino acid is transaminated in the presence of alpha-ketoglutarate to form oxaloacetate. The tritiated water is separated from the radiolabeled aspartate by passing the reaction mixture over a cation exchange column. Confirmation that the radioactivity in the product is associated with water was obtained by separating it by anion exchange HPLC and by evaporation. The product formation is linear with time up to 120 min and with tissue in the 0.05- to 10-micrograms range. The apparent Km for aspartate in the rat brain homogenate is found to be 0.83 mM and that for alpha-ketoglutarate to be 0.12 mM. Methods that further improve the sensitivity of the assay are also discussed.

  5. Resistance of horse alpha 1-proteinase inhibitor to perchloric acid denaturation and a simplified purification procedure resulting therefrom.

    PubMed

    Pellegrini, A; Hägeli, G; von Fellenberg, R

    1986-11-21

    Addition of perchloric acid (6.4% w/v final concentration) to horse alpha 1-proteinase inhibitor or to horse plasma neither precipitated nor inactivated alpha 1-proteinase inhibitor. None of the isoinhibitors of alpha 1-proteinase inhibitor was altered by dilute perchloric acid. This unexpected behavior led to a simplified procedure for the purification of horse alpha 1-proteinase inhibitor, consisting of removal of the bulk of plasma proteins, by perchloric acid precipitation and by gel filtration on Sephadex G-75 and G-200. The resulting preparations of alpha 1-proteinase inhibitor were immunogenically pure. The simplified purification procedure permitted the immunochemical comparison of the isoinhibitors of alpha 1-proteinase inhibitor, which proved to be immunologically identical. PMID:3022814

  6. Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei

    SciTech Connect

    Kutsyi, M.P.; Gaziev, A.I.

    1994-11-01

    An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after {gamma} irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiothreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats. 43 refs., 7 figs.

  7. Fourier transform interferometer alignment method.

    PubMed

    Goldberg, Kenneth A; Naulleau, Patrick; Bokor, Jeffrey

    2002-08-01

    A rapid and convenient method has been developed to facilitate the alignment of the image-plane components of point-diffraction interferometers, including the phase-shifting point-diffraction interferometer. In real time, the Fourier transform of the detected image is used to calculate a pseudoimage of the electric field in the image plane of the test optic where thecritical alignment o f variousoptical components is performed. Reconstruction of the pseudoimage is similar to off-axis, Fourier transform holography. Intermediate steps in the alignment procedure are described. Fine alignment is aided by the introduction and optimization of a global-contrast parameter that is easily calculated from the Fourier transform. Additional applications include the alignment of image-plane apertures in general optical systems, the rapid identification of patterned image-plane alignment marks, and the probing of important image-plane field properties. PMID:12153074

  8. New perspectives on basic mechanisms in lung disease. 6. Proteinase imbalance: its role in lung disease.

    PubMed Central

    Tetley, T D

    1993-01-01

    The hypothesis, some 30 years ago, that NE was the sole proteolytic agent responsible for the development of emphysema seems naive in retrospect. The availability of technology to measure NE facilitated the early research into the relationship between NE and lung disease. Despite an abundance of information on the activity of NE in the lung, it will probably require prospective studies in man with specific NE inhibitors or control at the gene level to establish a causal relationship between NE and lung disease. Parallel research has resulted in the isolation and characterisation of NE inhibitors other than PI and, indeed, alternative proteolytic enzymes that might contribute to lung disease. It is perhaps impossible now to think that a single proteinase, however omnipotent it may be, causes lung diseases as diverse as emphysema and fibrosis. An important aspect that is emerging is the interrelationship between proteolytic enzymes produced by different, or sometimes the same, cells that could potentiate tissue proteolysis. The evidence suggests that there is likely to be coordinated action between neutrophils, macrophages, and possibly mesenchymal proteinases which can activate or inactivate each other. In addition, one class of proteinases often appears able to proteolytically inactivate inhibitors of the opposite class, which presumably could amplify proteolysis if it occurred in vivo. Although the work on this aspect of proteinase activity is in its infancy, one suspects that part of the normal regulation of proteinase activity might include compartmentalisation. For example, the neutrophil stores proteinases before appropriate release and can inactivate PI to enable proteolytic action pericellularly, whereas degradation of extracellular matrix by macrophages requires interaction between the cell and matrix which is facilitated by cell receptor bound uPA. Disintegration of these "compartments" due to oedema, proteolysis, or for mechanical reasons could, firstly

  9. Static Fourier transform infrared spectrometer.

    PubMed

    Schardt, Michael; Murr, Patrik J; Rauscher, Markus S; Tremmel, Anton J; Wiesent, Benjamin R; Koch, Alexander W

    2016-04-01

    Fourier transform spectroscopy has established itself as the standard method for spectral analysis of infrared light. Here we present a robust and compact novel static Fourier transform spectrometer design without any moving parts. The design is well suited for measurements in the infrared as it works with extended light sources independent of their size. The design is experimentally evaluated in the mid-infrared wavelength region between 7.2 μm and 16 μm. Due to its large etendue, its low internal light loss, and its static design it enables high speed spectral analysis in the mid-infrared. PMID:27137061

  10. Role of saliva proteinase 3 in dental caries

    PubMed Central

    Yang, Teng-Yu; Zhou, Wen-Jie; Du, Yue; Wu, Song-Tao; Yuan, Wen-Wen; Yu, Yu; Su, Lin; Luo, Yang; Zhang, Jie-Hua; Lu, Wan-Lu; Wang, Xiao-Qian; Chen, Jiao; Feng, Yun; Zhou, Xue-Dong; Zhang, Ping

    2015-01-01

    Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson's correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL−1 or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3. PMID:26756046

  11. Potent and selective nonpeptidic inhibitors of procollagen C-proteinase.

    PubMed

    Fish, Paul V; Allan, Gillian A; Bailey, Simon; Blagg, Julian; Butt, Richard; Collis, Michael G; Greiling, Doris; James, Kim; Kendall, Jackie; McElroy, Andrew; McCleverty, Dawn; Reed, Charlotte; Webster, Robert; Whitlock, Gavin A

    2007-07-26

    6-Cyclohexyl-N-hydroxy-3-(1,2,4-oxadiazol-5-yl)hexanamides were previously disclosed as inhibitors of procollagen C-proteinase (PCP) culminating in the identification of amide 1. Our objective was to discover a second inhibitor that would have improved affinity for PCP and to optimize properties for transepidermal delivery (TED) to intact skin. Further investigation of this template identified a number of potent PCP inhibitors (IC50 values of 2-6 nM) with improved TED flux. Sulfonamide 56 had excellent PCP enzyme activity when measured with a peptide substrate (Ki 8.7 nM) or with the endogenous substrate procollagen (IC50 3.4 nM) and demonstrates excellent selectivity over MMPs involved in wound healing (>10 000-fold). In the fibroplasia model, 56 inhibited deposition of insoluble collagen by 76 +/- 2% at 10 microM and was very effective at penetrating human skin in vitro with a TED flux of 1.5 microg/cm2/h, which compares favorably with values for agents that are known to penetrate skin well in vivo. Based on this profile, 56 (UK-421,045) was selected as a candidate for further preclinical evaluation as a topically applied, dermal anti-scarring agent. PMID:17591762

  12. Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously.

    PubMed

    Gupta, Snehalata; Wang, Yang; Jiang, Haobo

    2005-03-01

    In the tobacco hornworm Manduca sexta, proteolytic activation of prophenoloxidase (proPO) is mediated by three proPO-activating proteinases (PAPs) and two serine proteinase homologs (SPHs) (Proceedings of the National Academy of Sciences, USA 95 (1998) 12220-12225; J. Biol. Chem. 278 (2003a) 3552-3561; Insect Biochem. Mol. Biol. 33 (2003b) 1049-1060). While our current data are consistent with the hypothesis that the SPHs serve as a cofactor/anchor for PAPs (Insect Biochemistry and Molecular Biology 33 (2003) 197-208; Insect Biochemistry and Molecular Biology 34 (2004) 731-742), roles of these clip-domain proteins (i.e. PAPs and SPHs) in proPO activation are poorly defined. To better understand this process, we further characterized the activation reaction using proPO, PAP-1 and SPHs. PAP-1 itself cleaved nearly 1/3 of proPO at Arg51 without generating much phenoloxidase (PO) activity. In the presence of SPHs, the cleavage of proPO became more complete while the increase in PO activity was over 20-fold, indicating that the extent of cleavage does not directly correlate with PO activity. Since SPHs and p-amidinophenyl methanesulfonyl fluoride (APMSF)-treated PAP-1 did not generate active PO by interacting with proPO, proteolytic cleavage is critical for proPO activation. After 1/5 of proPO was processed by PAP-1 alone which was then inactivated by M. sexta serpin-1J or APMSF, further incubation of the reaction mixture with SPHs failed to generate active PO either. Thus, SPHs cannot generate PO activity by simply binding to cleaved proPO. M. sexta proPO activation requires active PAP-1 and SPHs at the same time-one for limited proteolysis and the other as a cofactor, perhaps. Gel filtration chromatography and native gel electrophoresis revealed the PAP-SPH, proPO-PAP, and SPH-proPO associations, essential for generating high Mr, active PO at the site of infection. PMID:15705503

  13. Isolation and characterization of a serine proteinase specific to human C3b from human erythrocyte membranes

    SciTech Connect

    Charriaut, C.; Krikorian, L.; Barel, M.; Frade, R.

    1986-03-05

    In a previous report, they have shown that human C3b bound through CR1 to human erythrocytes is cleaved by a membrane proteinase activity. Following the molecular analysis of this proteinase activity, they have purified it by a four step procedure: ammonium sulfate precipitation, biogel filtration, fluid phase electrophoresis and hydroxylapatite chromatography. The highly purified proteinase was labeled by /sup 125/I iodine or /sup 3/H-DFP and analyzed by gel electrophoresis: a single band membrane component was characterized by its apparent molecular weight of 57 K or 60 K, under non reducing or reducing conditions respectively and was called p 57. Its reactivity with /sup 3/H-DFP and the inhibition by PMSF of the proteinase activity indicate that p 57 is a serine proteinase. Moreover, it is sensitive to aprotinin and ..gamma..1-antitrypsin. This membrane proteinase presents a higher activity in the presence of detergent and cleaves both alpha and beta chains of human C3b. Polyclonal antibody prepared against this purified proteinase inhibits its activity. On the basis of its structure and its functions, i.e. molecular weight, antigenic properties, proteinase properties and proteinases inhibitors sensitivity, p57 is not related to CR1 or DAF, two others membrane components which react with human C3b and identified by others on human erythrocytes. These specific antibodies allow to analyze the presence of p57 on human cells.

  14. Mutation of the Myxoma virus SERP2 P1-site to prevent proteinase inhibition causes apoptosis in cultured RK-13 cells and attenuates disease in rabbits, but mutation to alter specificity causes apoptosis without reducing virulence.

    PubMed

    MacNeill, Amy L; Turner, Peter C; Moyer, Richard W

    Myxoma virus (MYX) prevents apoptosis in RK-13 cells and forms thick dermal lesions with 100% mortality in rabbits. MYX encodes the virulence factor SERP2, a serine proteinase inhibitor (serpin). SERP2 was mutated to evaluate SERP2 function during MYX infection. MYXDeltaSERP2::lacZ (deleted for SERP2) did not inhibit apoptosis in RK-13 cells; infected rabbits had thin dermal lesions and <10% mortality. MYX-SERP2-D294A, a P1-site aspartate to alanine mutant, inactivated the serpin; infection was indistinguishable from MYXDeltaSERP2::lacZ. SERP2-D294E prevented inhibition of caspase-8, caspase-10 and granzyme-B; and MYX-SERP2-D294E failed to block apoptosis in RK-13 cells, but was fully virulent in rabbits. MYXDeltaSERP2::crmA expressed crmA instead of SERP2 and inhibited apoptosis in cell culture, but caused thin lesions and only 70% mortality in rabbits, hence crmA cannot fully substitute for SERP2. Control of apoptosis in culture does not correlate with virulence in rabbits. Virulence may instead depend on inhibition of proinflammatory proteinases by SERP2. PMID:16959285

  15. Fourier spectroscopy and planetary research

    NASA Technical Reports Server (NTRS)

    Hanel, R. A.; Kunde, V. G.

    1974-01-01

    The application of Fourier Transform Spectroscopy (FTS) to planetary research is reviewed. The survey includes FTS observations of the sun, all the planets except Uranus and Pluto, the Galilean satellites and Saturn's rings. Instrumentation and scientific results are considered and the prospects and limitations of FTS for planetary research in the forthcoming years are discussed.

  16. Fourier Analysis of Musical Intervals

    ERIC Educational Resources Information Center

    LoPresto, Michael C.

    2008-01-01

    Use of a microphone attached to a computer to capture musical sounds and software to display their waveforms and harmonic spectra has become somewhat commonplace. A recent article in "The Physics Teacher" aptly demonstrated the use of MacScope in just such a manner as a way to teach Fourier analysis. A logical continuation of this project is to…

  17. Fourier Lucas-Kanade algorithm.

    PubMed

    Lucey, Simon; Navarathna, Rajitha; Ashraf, Ahmed Bilal; Sridharan, Sridha

    2013-06-01

    In this paper, we propose a framework for both gradient descent image and object alignment in the Fourier domain. Our method centers upon the classical Lucas & Kanade (LK) algorithm where we represent the source and template/model in the complex 2D Fourier domain rather than in the spatial 2D domain. We refer to our approach as the Fourier LK (FLK) algorithm. The FLK formulation is advantageous when one preprocesses the source image and template/model with a bank of filters (e.g., oriented edges, Gabor, etc.) as 1) it can handle substantial illumination variations, 2) the inefficient preprocessing filter bank step can be subsumed within the FLK algorithm as a sparse diagonal weighting matrix, 3) unlike traditional LK, the computational cost is invariant to the number of filters and as a result is far more efficient, and 4) this approach can be extended to the Inverse Compositional (IC) form of the LK algorithm where nearly all steps (including Fourier transform and filter bank preprocessing) can be precomputed, leading to an extremely efficient and robust approach to gradient descent image matching. Further, these computational savings translate to nonrigid object alignment tasks that are considered extensions of the LK algorithm, such as those found in Active Appearance Models (AAMs). PMID:23599053

  18. Fourier Series and Elliptic Functions

    ERIC Educational Resources Information Center

    Fay, Temple H.

    2003-01-01

    Non-linear second-order differential equations whose solutions are the elliptic functions "sn"("t, k"), "cn"("t, k") and "dn"("t, k") are investigated. Using "Mathematica", high precision numerical solutions are generated. From these data, Fourier coefficients are determined yielding approximate formulas for these non-elementary functions that are…

  19. 40 CFR 721.10348 - Aspartic acid, N,N′-(iminodi-alkanediyl)bis, tetraalkane esters (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Aspartic acid, N,Nâ²-(iminodi... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10348 Aspartic acid, N,N′-(iminodi... reporting. (1) The chemical substances identified generically as aspartic acid,...

  20. 40 CFR 721.10348 - Aspartic acid, N,N′-(iminodi-alkanediyl)bis, tetraalkane esters (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Aspartic acid, N,Nâ²-(iminodi... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10348 Aspartic acid, N,N′-(iminodi... reporting. (1) The chemical substances identified generically as aspartic acid,...

  1. 40 CFR 721.10348 - Aspartic acid, N,N′-(iminodi-alkanediyl)bis, tetraalkane esters (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Aspartic acid, N,Nâ²-(iminodi... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10348 Aspartic acid, N,N′-(iminodi... reporting. (1) The chemical substances identified generically as aspartic acid,...

  2. Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus).

    PubMed

    Laycock, M V; Hirama, T; Hasnain, S; Watson, D; Storer, A C

    1989-10-15

    A new cysteine proteinase was isolated from the digestive juice of the American lobster (Homarus americanus). The enzyme was purified by a combination of affinity and ion-exchange chromatography and gel filtration. The cysteine proteinase accounted for 80% of the proteolytic activity in the lumen of the hepatopancreas. The most potent heavy-metal inhibitors were Hg, Cu, and Ag ions. Inhibition by organic proteinase inhibitors, including E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane] and activation of the enzyme by 2-mercaptoethanol and dithiothreitol are characteristic of cysteine proteinases. Several similarities to papain are noted and include the N-terminal sequence, of which 22 of the first 28 amino acids are identical. Some notable differences are the higher Mr of 28,000 compared with 23,350 for papain, and the low isoelectric point (pI 4.5) of the lobster enzyme. The effects of pH and temperature on catalytic activity of the lobster proteinase were studied with benzyloxycarbonylalanine p-nitrophenyl ester as the substrate. The kcat./Km value was effectively temperature-independent between 10 and 60 degrees C. The pH-activity profile for the lobster enzyme revealed four apparent protonation states, of which only two are active. PMID:2597115

  3. An alkaline serine-proteinase from a bacterium isolated from bat feces: purification and characterization.

    PubMed

    Tanskul, Somporn; Hiraga, Kazumi; Takada, Katsumi; Rungratchote, Suchart; Suntinanalert, Prasert; Oda, Kohei

    2009-11-01

    An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 degrees C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H(2)O(2), 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P(1) position, in descending order. The k(cat), K(m) and k(cat)/K(m) values for Z-Val-Lys-Met-MCA were 16.8+/-0.14 min(-1), 5.1+/-0.28 microM, and 3.3+/-0.28 microM(-1) min(-1) respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS. PMID:19897920

  4. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  5. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi.

    PubMed

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-09-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  6. Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

    PubMed Central

    Böckle, B; Galunsky, B; Müller, R

    1995-01-01

    A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%. PMID:7487006

  7. Proteinase-antiproteinase balance in tracheal aspirates from neonates.

    PubMed

    Sluis, K B; Darlow, B A; Vissers, M C; Winterbourn, C C

    1994-02-01

    We wanted to identify the inhibitors of neutrophil elastase, quantify their activities in the upper airways of neonates, and relate these to the presence of active elastase and the likelihood of elastolytic injury occurring due to inhibitory capacity being overwhelmed. Activities of neutrophil elastase and its inhibitors were measured in tracheal aspirates from 17 infants, 10 of whom subsequently developed bronchopulmonary dysplasia. All aspirates contained immunologically detectable alpha 1-proteinase inhibitor (alpha 1-PI), but their inhibitory capacity against neutrophil elastase ranged from being undetectable to being in excess of the amount of alpha 1-PI detected immunologically. When the alpha 1-PI was removed from each of the aspirates, using a specific antibody, from 0-50% of the original activity remained, indicating the presence of another elastase inhibitor. Its properties were consistent with it being the low molecular mass, secretory leucoproteinase inhibitor (SLPI), also known as bronchial antileucoproteinase. The alpha 1-PI was from 0-100% active. Most of the inactive inhibitor was shown by western blotting to be complexed with elastase, with a small amount of cleaved material. There was no evidence of major oxidative inactivation. Free elastase was detected in only three of the aspirates; these had little or no detectable elastase inhibitory capacity, and most of their alpha 1-PI was complexed. Elastase load, comprising the sum of free and complexed elastase, correlated closely with myeloperoxidase activity, a recognized marker of inflammatory activity. Active SLPI levels showed a positive correlation with gestational age (r = 0.66). We conclude that most neutrophil elastase in the upper airways of ventilated infants is complexed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7909297

  8. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    SciTech Connect

    Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus; Calafat, Jero; Janssen, Hans; Persson, Ann-Maj; Hansson, Markus; Olsson, Inge

    2010-11-15

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  9. Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

    PubMed Central

    Boudier, C; Bieth, J G

    1994-01-01

    N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not. PMID:7945266

  10. Compact Microwave Fourier Spectrum Analyzer

    NASA Technical Reports Server (NTRS)

    Savchenkov, Anatoliy; Matsko, Andrey; Strekalov, Dmitry

    2009-01-01

    A compact photonic microwave Fourier spectrum analyzer [a Fourier-transform microwave spectrometer, (FTMWS)] with no moving parts has been proposed for use in remote sensing of weak, natural microwave emissions from the surfaces and atmospheres of planets to enable remote analysis and determination of chemical composition and abundances of critical molecular constituents in space. The instrument is based on a Bessel beam (light modes with non-zero angular momenta) fiber-optic elements. It features low power consumption, low mass, and high resolution, without a need for any cryogenics, beyond what is achievable by the current state-of-the-art in space instruments. The instrument can also be used in a wide-band scatterometer mode in active radar systems.

  11. Fourier analysis of blazar variability

    SciTech Connect

    Finke, Justin D.; Becker, Peter A.

    2014-08-10

    Blazars display strong variability on multiple timescales and in multiple radiation bands. Their variability is often characterized by power spectral densities (PSDs) and time lags plotted as functions of the Fourier frequency. We develop a new theoretical model based on the analysis of the electron transport (continuity) equation, carried out in the Fourier domain. The continuity equation includes electron cooling and escape, and a derivation of the emission properties includes light travel time effects associated with a radiating blob in a relativistic jet. The model successfully reproduces the general shapes of the observed PSDs and predicts specific PSD and time lag behaviors associated with variability in the synchrotron, synchrotron self-Compton, and external Compton emission components, from submillimeter to γ-rays. We discuss applications to BL Lacertae objects and to flat-spectrum radio quasars (FSRQs), where there are hints that some of the predicted features have already been observed. We also find that FSRQs should have steeper γ-ray PSD power-law indices than BL Lac objects at Fourier frequencies ≲ 10{sup –4} Hz, in qualitative agreement with previously reported observations by the Fermi Large Area Telescope.

  12. Subharmonic Fourier domain mode locking.

    PubMed

    Eigenwillig, Christoph M; Wieser, Wolfgang; Biedermann, Benjamin R; Huber, Robert

    2009-03-15

    We demonstrate a subharmonically Fourier domain mode-locked wavelength-swept laser source with a substantially reduced cavity fiber length. In contrast to a standard Fourier domain mode-locked configuration, light is recirculated repetitively in the delay line with the optical bandpass filter used as switch. The laser has a fundamental optical round trip frequency of 285 kHz and can be operated at integer fractions thereof (subharmonics). Sweep ranges up to 95 nm full width centred at 1317 nm are achieved at the 1/5th subharmonic. A maximum sensitivity of 116 dB and an axial resolution of 12 microm in air are measured at an average sweep power of 12 mW. A sensitivity roll-off of 11 dB over 4 mm and 25 dB over 10 mm is observed and optical coherence tomography imaging is demonstrated. Besides the advantage of a reduced fiber length, subharmonic Fourier domain mode locking (shFDML) enables simple scaling of the sweep speed by extracting light from the delay part of the resonator. A sweep rate of 570 kHz is achieved. Characteristic features of shFDML operation, such as power leakage during fly-back and cw breakthrough, are investigated. PMID:19282912

  13. Cleavage of fibrinogen by proteinases elicits allergic responses through Toll-like receptor 4.

    PubMed

    Millien, Valentine Ongeri; Lu, Wen; Shaw, Joanne; Yuan, Xiaoyi; Mak, Garbo; Roberts, Luz; Song, Li-Zhen; Knight, J Morgan; Creighton, Chad J; Luong, Amber; Kheradmand, Farrah; Corry, David B

    2013-08-16

    Proteinases and the innate immune receptor Toll-like receptor 4 (TLR4) are essential for expression of allergic inflammation and diseases such as asthma. A mechanism that links these inflammatory mediators is essential for explaining the fundamental basis of allergic disease but has been elusive. Here, we demonstrate that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen, yielding fibrinogen cleavage products that acted as TLR4 ligands on airway epithelial cells and macrophages. Thus, allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation. These findings clarify the molecular basis of allergic disease and suggest new therapeutic strategies. PMID:23950537

  14. Thiol-activated serine proteinases from nymphal hemolymph of the African migratory locust, Locusta migratoria migratorioides.

    PubMed

    Hanzon, Jacob; Smirnoff, Patricia; Applebaum, Shalom W; Mattoo, Autar K; Birk, Yehudith

    2003-02-01

    Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds. PMID:12559979

  15. The anthelmintic efficacy of natural plant cysteine proteinases against Hymenolepis microstoma in vivo.

    PubMed

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2015-09-01

    Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections in vivo. Hymenolepis microstoma is a natural parasite of house mice, and provides a convenient model system for the assessment of novel drugs for anthelmintic activity against cestodes. The experiments described in this paper indicate that treatment of H. microstoma infections in mice with the supernatant of papaya latex (PLS), containing active cysteine proteinases, is only minimally efficacious. The statistically significant effects seen on worm burden and biomass showed little evidence of dose dependency, were temporary and the role of cysteine proteinases as the active principles in PLS was not confirmed by specific inhibition with E-64. Worm fecundity was not affected by treatment at the doses used. We conclude also that this in vivo host-parasite system is not sensitive enough to be used reliably for the detection of cestocidal activity of compounds being screened as potential, novel anthelmintics. PMID:25226116

  16. Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae).

    PubMed

    Naveh, V Hosseini; Bandani, A R; Azmayeshfard, P; Hosseinkhani, S

    2005-01-01

    The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases. PMID:16628932

  17. Salicylic Acid Inhibits Synthesis of Proteinase Inhibitors in Tomato Leaves Induced by Systemin and Jasmonic Acid.

    PubMed Central

    Doares, S. H.; Narvaez-Vasquez, J.; Conconi, A.; Ryan, C. A.

    1995-01-01

    Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol Mol Plant Pathol 33: 377-384), and linolenic acid (H. Pena-Cortes, T. Albrecht, S. Prat, E.W. Weiler, L. Willmitzer [1993] Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step in the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes. PMID:12228577

  18. Carboxy-terminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases.

    PubMed

    Michaud, D; Cantin, L; Vrain, T C

    1995-10-01

    The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a approximately 10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearance of OCIIi resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control. PMID:7574723

  19. [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

  20. Occurrence of Free d-Amino Acids and Aspartate Racemases in Hyperthermophilic Archaea

    PubMed Central

    Matsumoto, Megumi; Homma, Hiroshi; Long, Zhiqun; Imai, Kazuhiro; Iida, Toshii; Maruyama, Tadashi; Aikawa, Yuko; Endo, Isao; Yohda, Masafumi

    1999-01-01

    The occurrence of free d-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of d-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70°C, and aspartate racemase homologous genes in them were identified by PCR. d-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in Thermococcus strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that d-amino acids are produced in some hyperthermophilic archaea, although their function is unknown. PMID:10515953

  1. Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995

    SciTech Connect

    Dougherty, W.

    1995-10-01

    The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function in a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.

  2. Purification and Characterization of an Endophytic Fungal Proteinase That Is Abundantly Expressed in the Infected Host Grass.

    PubMed Central

    Lindstrom, J. T.; Belanger, F. C.

    1994-01-01

    A novel Acremonium typhinum proteinase that is expressed during endophytic infection of the grass Poa ampla Merr. was purified from endophyte-infected leaf sheath tissue. It is a thiol-containing serine alkaline endoproteinase with bound carbohydrate. In the infected host tissue, this proteinase is an abundant protein localized within fungal membrane vesicles and in the plant and/or fungal cell walls. This proteinase was not expressed constitutively during fungus culture. Rather, its expression appeared to be induced by nutrient depletion. Expression of an antigenically similar proteinase was detected in five other endophyte-infected Poa species. The regulated expression of the proteinase in culture and its abundance in infected plant tissue suggest that its expression may be involved in the symbiotic interaction of the plant and the fungus. PMID:12232300

  3. Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

    SciTech Connect

    Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

    1988-06-01

    E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

  4. IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes.

    PubMed

    von Pawel-Rammingen, Ulrich; Björck, Lars

    2003-02-01

    The Gram-positive bacterium Streptococcus pyogenes is a major human pathogen causing substantial morbidity and mortality in society. S. pyogenes has evolved numerous molecular mechanisms to avoid the various actions of the human immune system and has established means to modulate both adaptive and innate immune responses. S. pyogenes produces and secretes proteolytic enzymes, which have an important impact on the ability of the bacteria to survive in the human host. Prominent among these are two immunoglobulin-degrading enzymes: the newly discovered streptococcal cysteine proteinase, IdeS, and the classical cysteine proteinase of S. pyogenes, SpeB. PMID:12615219

  5. Unusual chromatographic behaviour and one-step purification of a novel membrane proteinase from Bacillus cereus.

    PubMed

    Fricke, B; Buchmann, T; Friebe, S

    1995-11-01

    Cell envelopes of Bacillus cereus contain a casein-cleaving membrane proteinase (CCMP) and an insulin-cleaving membrane proteinase (ICMP), which differ in their substrate and inhibitor specificity from all Bacillus proteinases described previously. They remained localized in the cytoplasmic membrane after treatment with lysozyme and mutanolysin and they are strongly attached to the membrane compared with other known membrane proteinases. Only high a concentration of the Zwitterionic detergent sulfobetain SB-12 enabled an effective solubilization of both membrane proteinases. The usual conventional purification methods, such as chromatofocusing, ion-exchange chromatography and hydrophobic interaction chromatography in the presence of detergent concentrations beyond their critical micelle concentration, could not be applied to the purification, because the solubilized membrane proteinases bound strongly and irreversibly to the chromatographic matrix. In the search for other purification methods, we used a tentacle ion-exchanger (EMD trimethylaminoethyl-Fractogel) to reduce the hydrophobic interactions between the proteinases and the matrix. All contaminating proteins could be removed by a first gradient of sodium chloride without elution of CCMP; a second gradient with isopropanol and a decreasing salt concentration resulted in an efficiently purified CCMP. The ICMP was irreversibly denaturated. Purified CCMP is a member of the metalloproteinase family with a pH optimum in the neutral range and a temperature optimum of 40 degrees C, whose properties differ from the serine-type membrane proteinase of Bacillus subtilis described by Shimizu et al. [Agric. Biol. Chem., 47 (1983) 1775]. It consists of two subunits in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Mr 53,000 and 65,000); however, the molecular mass of the purified enzyme could not be determined by size exclusion or SDS-PAGE, because the purified enzyme

  6. Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis.

    PubMed

    Coutinho, S D; Paula, C R

    2000-02-01

    The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin-sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform. PMID:10746230

  7. Expression of human α1-proteinase inhibitor in Aspergillus niger

    PubMed Central

    Karnaukhova, Elena; Ophir, Yakir; Trinh, Loc; Dalal, Nimish; Punt, Peter J; Golding, Basil; Shiloach, Joseph

    2007-01-01

    Background Human α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high

  8. A Simple Approach to Fourier Aliasing

    ERIC Educational Resources Information Center

    Foadi, James

    2007-01-01

    In the context of discrete Fourier transforms the idea of aliasing as due to approximation errors in the integral defining Fourier coefficients is introduced and explained. This has the positive pedagogical effect of getting to the heart of sampling and the discrete Fourier transform without having to delve into effective, but otherwise long and…

  9. Aperture scanning Fourier ptychographic microscopy

    PubMed Central

    Ou, Xiaoze; Chung, Jaebum; Horstmeyer, Roarke; Yang, Changhuei

    2016-01-01

    Fourier ptychographic microscopy (FPM) is implemented through aperture scanning by an LCOS spatial light modulator at the back focal plane of the objective lens. This FPM configuration enables the capturing of the complex scattered field for a 3D sample both in the transmissive mode and the reflective mode. We further show that by combining with the compressive sensing theory, the reconstructed 2D complex scattered field can be used to recover the 3D sample scattering density. This implementation expands the scope of application for FPM and can be beneficial for areas such as tissue imaging and wafer inspection. PMID:27570705

  10. Fourier-transform optical microsystems

    NASA Technical Reports Server (NTRS)

    Collins, S. D.; Smith, R. L.; Gonzalez, C.; Stewart, K. P.; Hagopian, J. G.; Sirota, J. M.

    1999-01-01

    The design, fabrication, and initial characterization of a miniature single-pass Fourier-transform spectrometer (FTS) that has an optical bench that measures 1 cm x 5 cm x 10 cm is presented. The FTS is predicated on the classic Michelson interferometer design with a moving mirror. Precision translation of the mirror is accomplished by microfabrication of dovetailed bearing surfaces along single-crystal planes in silicon. Although it is miniaturized, the FTS maintains a relatively high spectral resolution, 0.1 cm-1, with adequate optical throughput.

  11. JPL Fourier transform ultraviolet spectrometer

    NASA Technical Reports Server (NTRS)

    Cageao, R. P.; Friedl, R. R.; Sander, Stanley P.; Yung, Y. L.

    1994-01-01

    The Fourier Transform Ultraviolet Spectrometer (FTUVS) is a new high resolution interferometric spectrometer for multiple-species detection in the UV, visible and near-IR. As an OH sensor, measurements can be carried out by remote sensing (limb emission and column absorption), or in-situ sensing (long-path absorption or laser-induced fluorescence). As a high resolution detector in a high repetition rate (greater than 10 kHz) LIF system, OH fluorescence can be discriminated against non-resonant background emission and laser scatter, permitting (0, 0) excitation.

  12. Fourier-Transform Infrared Spectrometer

    NASA Technical Reports Server (NTRS)

    Schindler, R. A.

    1986-01-01

    Fourier-transform spectrometer provides approximately hundredfold increase in luminosity at detector plane over that achievable with older instruments of this type. Used to analyze such weak sources as pollutants and other low-concentration substances in atmosphere. Interferometer creates fringe patterns on two distinct arrays of light detectors, which observe different wavelength bands. Objective lens focuses scene on image plane, which contains optical chopper. To make instrument less susceptible to variations in scene under observation, field and detector lenses focus entrance aperture, rather that image, onto detector array.

  13. Improved Fourier-transform profilometry

    SciTech Connect

    Mao Xianfu; Chen Wenjing; Su Xianyu

    2007-02-10

    An improved optical geometry of the projected-fringe profilometry technique, in which the exit pupil of the projecting lens and the entrance pupil of the imaging lens are neither at the same height above the reference plane nor coplanar, is discussed and used in Fourier-transform profilometry. Furthermore, an improved fringe-pattern description and phase-height mapping formula based on the improved geometrical generalization is deduced. Employing the new optical geometry, it is easier for us to obtain the full-field fringe by moving either the projector or the imaging device. Therefore the new method offers a flexible way to obtain reliable height distribution of a measured object.

  14. Crystal structure of truncated aspartate transcarbamoylase from Plasmodium falciparum.

    PubMed

    Lunev, Sergey; Bosch, Soraya S; Batista, Fernando de Assis; Wrenger, Carsten; Groves, Matthew R

    2016-07-01

    The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure of PfATC is reported. The space group of the PfATC crystals was determined to be monoclinic P21, with unit-cell parameters a = 87.0, b = 103.8, c = 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presented PfATC model shares a high degree of homology with the catalytic domain of Escherichia coli ATC. There is as yet no evidence of the existence of a regulatory domain in PfATC. Similarly to E. coli ATC, PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described. PMID:27380369

  15. FFTLog: Fast Fourier or Hankel transform

    NASA Astrophysics Data System (ADS)

    Hamilton, Andrew J. S.

    2015-12-01

    FFTLog is a set of Fortran subroutines that compute the fast Fourier or Hankel (= Fourier-Bessel) transform of a periodic sequence of logarithmically spaced points. FFTLog can be regarded as a natural analogue to the standard Fast Fourier Transform (FFT), in the sense that, just as the normal FFT gives the exact (to machine precision) Fourier transform of a linearly spaced periodic sequence, so also FFTLog gives the exact Fourier or Hankel transform, of arbitrary order m, of a logarithmically spaced periodic sequence.

  16. Pediatric anti-N methyl D aspartate receptor encephalitis.

    PubMed

    Suri, Vinit; Sharma, Sushma; Gupta, Rohan; Sogani, S K; Mediratta, Sunit; Jadhao, Nilesh

    2013-05-01

    Anti-N Methyl D Aspartate Receptor encephalitis (anti-NMDARE) is a recently defined disease, which is probably more under-recognized than rare. We report a case of anti-NMDARE in a 13-years-old girl, who presented with intractable seizures. To the best of our knowledge, this is the second case of pediatric anti-NMDARE being reported from India. The need for a greater awareness of this disease and the subtle differences in clinical presentation between pediatric and adult patients are highlighted. PMID:24082929

  17. High-molecular-mass multicatalytic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR.

    PubMed Central

    Benoist, P; Müller, A; Diem, H G; Schwencke, J

    1992-01-01

    A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them. Images PMID:1537794

  18. Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

    PubMed

    Aihara, Takayuki; Ito, Toshiya; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Sekine, Masae; Homma, Hiroshi; Yohda, Masafumi

    2016-07-01

    Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus. PMID:27094682

  19. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  20. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    PubMed

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions. PMID:25462979

  1. Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

  2. Proteinase K and the structure of PrPse: the good, the bad, and the ugly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunod...

  3. LEKTI domain 15 is a functional Kazal-type proteinase inhibitor.

    PubMed

    Vitzithum, Klaus; Lauber, Thomas; Kreutzmann, Peter; Schulz, Axel; Sommerhoff, Christian P; Rösch, Paul; Marx, Ute C

    2008-01-01

    The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1' site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family. PMID:17936012

  4. Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis.

    PubMed

    Csoma, C; Polgár, L

    1984-09-15

    To degrade storage proteins germinating seeds synthesize proteinases de novo that can be inhibited by thiol-blocking reagents [Baumgartner & Chrispeels (1977) Eur. J. Biochem. 77, 223-233]. We have elaborated a procedure for isolation of such a proteinase from the cotyledons of Phaseolus vulgaris. The purification procedure involved fractionation of the cotyledon homogenate with acetone and with (NH4)2SO4 and successive chromatographies on DEAE-cellulose, activated thiol-Sepharose Sepharose and Sephacryl S-200. The purified enzyme has an Mr of 23,400, proved to be highly specific for the asparagine side chain and blocking of its thiol group resulted in loss of the catalytic activity. The chemical properties of the thiol group of the bean enzyme were investigated by acylation with t-butyloxycarbonyl-L-asparagine p-nitro-phenyl ester and by alkylations with iodoacetamide and iodoacetate. Deviations from normal pH-rate profile were observed, which indicated that the thiol group is not a simple functional group, but constitutes a part of an interactive system at the active site. The pKa value for acylation and the magnitude of the rate constant for alkylation with iodoacetate revealed that the bean proteinase possesses some properties not shared by papain and the other cysteine proteinases studied to date. PMID:6385962

  5. Nitric oxide inhibits cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi.

    PubMed

    Venturini, G; Salvati, L; Muolo, M; Colasanti, M; Gradoni, L; Ascenzi, P

    2000-04-13

    Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences. PMID:10753643

  6. OZONE EFFECTS ON ALPHA-1-PROTEINASE INHIBITOR IN VIVO: BLOOD PLASMA INHIBITORY ACTIVITY IS UNCHANGED

    EPA Science Inventory

    The possible oxidative inactivation of human blood plasma alpha-1-proteinase inhibitor (PI) by inhaled ozone was assessed. Eleven male volunteers (non-smokers) were exposed to 0.5 ppm ozone for four hours on two consecutive days and ten control subjects were exposed to air under ...

  7. Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis.

    PubMed Central

    Imamura, T; Potempa, J; Pike, R N; Travis, J

    1995-01-01

    Infection with Porphyromonas gingivalis is strongly associated with adult periodontitis, and proteinases are considered to be important virulent factors of the bacterium. In order to investigate the function of proteinases in disease development we examined vesicles, a biological carrier of these enzymes, for the generation of vascular permeability enhancement (VPE) activity, believed to correlate with the exudation of gingival crevicular fluid. The vesicles generated VPE activity from human plasma in a dose-dependent manner which could be inhibited 90% by antipain, a specific inhibitor of the Arg-specific cysteine proteinases (Arg-gingipains [RGPs] from P. gingivalis. Incubation of vesicles with high-molecular-weight-kininogen (HMWK)-deficient plasma did not result in VPE activity. On this basis, RGPs associated with vesicles were assumed to be responsible for most of the VPE activity generation via plasma prekallikrein activation and subsequent bradykinin production. The secondary pathway for VPE activity production was dependent on the direct release of bradykinin from HMWK by the concerted action of RGP and a Lys-specific cysteine proteinase (Lys-gingipain [KGP]), also associated with vesicles. These results indicate that RGP and KGP are biologically important VPE factors acting either via prekallikrein activation (RGP) and/or HMWK cleavage (RGP and KGP) to release BK and, thereby, contributing to the production of gingival crevicular fluid at periodontal sites infected with P. gingivalis. PMID:7729914

  8. MYOCARDIAL NECROSIS PRODUCED IN ANIMALS BY MEANS OF CRYSTALLINE STREPTOCOCCAL PROTEINASE

    PubMed Central

    Kellner, Aaron; Robertson, Theodore

    1954-01-01

    Focal myocardial necrosis that was often extensive was found in a high percentage of rabbits, guinea pigs, and mice given a single intravenous injection of crystalline streptococcal proteinase. The findings are discussed in relation to their possible implications for the pathogenesis of the cardiac lesions of rheumatic fever. PMID:13163324

  9. Nutritional requirements and nitrogen-dependent regulation of proteinase activity of Lactobacillus helveticus CRL 1062.

    PubMed

    Hebert, E M; Raya, R R; De Giori, G S

    2000-12-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or beta-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%. PMID:11097908

  10. Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

    PubMed

    Rockey, D D; Turaga, P S; Wiens, G D; Cook, B A; Kaattari, S L

    1991-10-01

    Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol). PMID:1777853

  11. Isolation and characterization of a proteinase K sensitive PrPSc fraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have shown that a sizeable fraction of PrPSc present in prion-infected tissues is,contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in att...

  12. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors.

    PubMed

    Turk, B; Turk, V; Turk, D

    1997-01-01

    Cysteine proteinases are widely distributed among living organisms. According to the most recent classifications (Rawlings and Barrett, 1993, 1994), they can be subdivided on the basis of sequence homology into 14 or even 20 different families, the most important being the papain and the calpain families. The papain-like cysteine proteinases are the most abundant among the cysteine proteinases. The family consists of papain and related plant proteinases such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, and the lysosomal cathepsins B, H, L, S, C and K. Most of these enzymes are relatively small proteins with Mr values in the range 20000-35000 (reviewed in Brocklehurst et al., 1987; Polgar, 1989; Rawlings and Barrett, 1994; Berti and Storer, 1995), with the exception of cathepsin C, which is an oligomeric enzyme with Mr approximately 200000 (Metrione et al., 1970; Dolenc et al., 1995). A number of cysteine proteinases are located within lysosomes. Four of them, cathepsins B, C, H and L, are ubiquitous in lysosomes of animals, whereas cathepsin S has a more restricted localisation (Barrett and Kirschke, 1981; Kirschke and Wiederanders, 1994). The enzymes, except cathepsin C, are endopeptidases (reviewed in Kirschke et al., 1995), although cathepsin B was found also to be a dipeptidyl carboxypeptidase (Aronson and Barrett, 1978) and cathepsin H also an aminopeptidase (Koga et al., 1992). Cathepsin C is a dipeptidyl aminopeptidase, but at higher pH it exhibits also dipeptidyl transferase activity (reviewed in Kirschke et al., 1995). Among the lysosomal cysteine proteinases, cathepsin L was found to be the most active in degradation of protein substrates, such as collagen, elastin and azocasein (Barrett and Kirschke, 1981; Maciewicz et al., 1987; Mason et al., 1989), arid cathepsin B the most abundant (Kirschke and Barrett, 1981). All the enzymes are optimally active at slightly acidic pH, although their pH optima for degradation of synthetic

  13. Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis.

    PubMed Central

    Jagels, M A; Travis, J; Potempa, J; Pike, R; Hugli, T E

    1996-01-01

    The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at

  14. Enzymatic response of the eucalypt defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a bis-benzamidine proteinase Inhibitor. i.

    PubMed

    Marinho-Prado, Jeanne Scardini; Lourenção, A L; Guedes, R N C; Pallini, A; Oliveira, J A; Oliveira, M G A

    2012-10-01

    Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition. PMID:23950094

  15. A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.

    PubMed

    Xie, Jian; Owen, Timothy; Xia, Ke; Callahan, Brian; Wang, Chunyu

    2016-08-31

    Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ∼2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation. PMID:27529645

  16. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    PubMed

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin. PMID:27108177

  17. Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

    PubMed Central

    Koehler, S M; Ho, T H

    1990-01-01

    Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression. PMID:2152126

  18. Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat.

    PubMed

    Wilson, Michael J; Lind, Jeremy; Sinha, Akhouri A

    2015-08-01

    Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not

  19. Digital pathology with Fourier ptychography

    PubMed Central

    Horstmeyer, Roarke; Ou, Xiaoze; Zheng, Guoan; Willems, Phil; Yang, Changhuei

    2015-01-01

    Fourier ptychographic microscopy (FPM) is a recently introduced method of acquiring high-resolution, wide field of view (FOV) giga-pixel histology images. The FPM procedure first acquires a sequence of low-resolution images of a sample under variable-angle illumination. It then combines these images using a novel phase retrieval algorithm to improve the employed microscope’s resolution beyond its conventional limit. Here, we first describe how FPM’s resolution improvement can enhance wide FOV histology imaging. Second, we show that FPM also records a thin sample’s optical phase, which can help pathologists digitally extract as much information as possible from a given histology slide. PMID:25481664

  20. Biosynthesis of D-aspartate in mammals: the rat and human homologs of mouse aspartate racemase are not responsible for the biosynthesis of D-aspartate.

    PubMed

    Matsuda, Satsuki; Katane, Masumi; Maeda, Kazuhiro; Kaneko, Yuusuke; Saitoh, Yasuaki; Miyamoto, Tetsuya; Sekine, Masae; Homma, Hiroshi

    2015-05-01

    D-Aspartate (D-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of D-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the D-Asp content and the expression levels of the mRNAs encoding GOT1L1 and D-Asp oxidase, a primary degradative enzyme of D-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on D-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in D-Asp biosynthesis. Therefore, D-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved. PMID:25646960

  1. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    PubMed

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that

  2. Fourier's law: insight from a simple derivation.

    PubMed

    Dubi, Y; Di Ventra, M

    2009-04-01

    The onset of Fourier's law in a one-dimensional quantum system is addressed via a simple model of weakly coupled quantum systems in contact with thermal baths at their edges. Using analytical arguments we show that the crossover from the ballistic (invalid Fourier's law) to diffusive (valid Fourier's law) regimes is characterized by a thermal length scale, which is directly related to the profile of the local temperature. In the same vein, dephasing is shown to give rise to classical Fourier's law, similarly to the onset of Ohm's law in mesoscopic conductors. PMID:19518279

  3. Aspartate aminotransferase activity in human healthy and inflamed dental pulps.

    PubMed

    Spoto, G; Fioroni, M; Rubini, C; Tripodi, D; Perinetti, G; Piattelli, A

    2001-06-01

    Aspartate aminotransferase (AST) seems to be an important mediator of inflammatory processes. Its role in the progression and detection of inflammatory periodontal disease has been increasingly recognized in recent years. In the present study AST activity was analyzed in normal healthy human dental pulps, in reversible pulpitis, and in irreversible pulpitis. Enzymatic AST activity showed that the control values for the healthy pulps were 4.8 +/- 0.7 units/mg of pulp tissue. In reversible pulpitis specimens the AST activity increased to 7.98 +/- 2.1 units/mg of pulp tissue. In irreversible pulpitis specimens the values decreased to 2.28 +/- 1.7 units/mg of pulp tissue. Differences between the groups (control versus reversible pulpitis and reversible pulpitis versus irreversible pulpitis) were statistically significant (p = 0.0015). These results could point to a role of AST in the early events that lead to development of pulpal inflammation. PMID:11487132

  4. Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

    PubMed

    Wales, M E; Madison, L L; Glaser, S S; Wild, J R

    1999-12-17

    The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity

  5. AGC1/2, the mitochondrial aspartate-glutamate carriers.

    PubMed

    Amoedo, N D; Punzi, G; Obre, E; Lacombe, D; De Grassi, A; Pierri, C L; Rossignol, R

    2016-10-01

    In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27132995

  6. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

    PubMed Central

    Abrahamson, M; Wikström, M; Potempa, J; Renvert, S; Hall, A

    1997-01-01

    AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in

  7. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3.

    PubMed Central

    Lobigs, M

    1993-01-01

    Flavivirus protein biosynthesis involves the proteolytic processing of a single polyprotein precursor by host- and virus-encoded proteinases. In this study, the requirement for the proteolytic function of the viral proteinase NS3 for correct processing of a polyprotein segment encompassing the Murray Valley encephalitis virus structural proteins is shown. The NS3-mediated cleavage in the structural polyprotein region presumably releases the capsid protein from its membrane anchor and triggers the appearance of the premembrane (prM) protein. This suggests that cleavage of prM by signal peptidase in the lumen of the endoplasmic reticulum is under control of a cytoplasmic cleavage catalyzed by a viral proteinase. The function of the viral proteinase is also essential for secretion of flaviviral spike proteins when expressed from cDNA via vaccinia virus recombinants or in COS cell transfections. This has important implications for the design of flavivirus subunit vaccines. Images Fig. 1 Fig. 2 Fig. 3 PMID:8392191

  8. Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.

    PubMed

    Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

    1996-08-01

    The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

  9. Fourier analysis and synthesis tomography.

    SciTech Connect

    Wagner, Kelvin H.; Sinclair, Michael B.; Feldkuhn, Daniel

    2010-05-01

    Most far-field optical imaging systems rely on a lens and spatially-resolved detection to probe distinct locations on the object. We describe and demonstrate a novel high-speed wide-field approach to imaging that instead measures the complex spatial Fourier transform of the object by detecting its spatially-integrated response to dynamic acousto-optically synthesized structured illumination. Tomographic filtered backprojection is applied to reconstruct the object in two or three dimensions. This technique decouples depth-of-field and working-distance from resolution, in contrast to conventional imaging, and can be used to image biological and synthetic structures in fluoresced or scattered light employing coherent or broadband illumination. We discuss the electronically programmable transfer function of the optical system and its implications for imaging dynamic processes. Finally, we present for the first time two-dimensional high-resolution image reconstructions demonstrating a three-orders-of-magnitude improvement in depth-of-field over conventional lens-based microscopy.

  10. Fourier transform infrared (FTIR) spectroscopy.

    PubMed

    Berthomieu, Catherine; Hienerwadel, Rainer

    2009-01-01

    Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method. PMID:19513810

  11. Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds.

    PubMed

    Hansen, Alexandar L; Behrman, Edward J

    2016-08-01

    We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid. PMID:27258673

  12. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    PubMed

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  13. Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

    PubMed

    Palmieri, L; Pardo, B; Lasorsa, F M; del Arco, A; Kobayashi, K; Iijima, M; Runswick, M J; Walker, J E; Saheki, T; Satrústegui, J; Palmieri, F

    2001-09-17

    The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle. PMID:11566871

  14. Properties of Copolymers of Aspartic Acid and Aliphatic Dicarboxylic Acids Prepared by Reactive Extrusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartic acid may be prepared chemically or by the fermentation of carbohydrates. Currently, low molecular weight polyaspartic acids are prepared commercially by heating aspartic acid at high temperatures (greater than 220 degrees C) for several hours in the solid state. In an effort to develop a ...

  15. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.

    PubMed

    Michaud, D; Cantin, L; Raworth, D A; Vrain, T C

    1996-01-01

    A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans. PMID:8907521

  16. 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

  17. Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation.

    PubMed Central

    Bingle, L.; Tetley, T. D.

    1996-01-01

    Secretory leukoprotease inhibitor (SLPI) is a low molecular weight serine proteinase inhibitor, notably of neutrophil elastase (NE), which is synthesised and secreted by the pulmonary epithelium. SLPI plays an important role in limiting NE-induced pulmonary inflammation and, significantly, it also possesses anti-HIV activity. SLPI is a significant component of the anti-NE shield in the lung which has different reactivity from, and is therefore complementary to, the anti-NE action of alpha 1-proteinase inhibitor (alpha 1-PI). Inhaled recombinant SLPI (rSLPI) could prove beneficial in partnership with alpha 1-PI in the treatment of a number of inflammatory lung disorders including emphysema, chronic bronchitis, cystic fibrosis, and adult respiratory distress syndrome. PMID:8994529

  18. When activity requires breaking up: LEKTI proteolytic activation cascade for specific proteinase inhibition.

    PubMed

    Furio, Laetitia; Hovnanian, Alain

    2011-11-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain proteinase inhibitor whose defective expression causes Netherton syndrome (NS). LEKTI is encoded by SPINK5, which is also a susceptibility gene for atopic disease. In this issue, Fortugno et al. report an elegant and thorough study of the LEKTI proteolytic activation process in which they identify the precise nature of the cleavage sites used and the bioactive fragments generated. They propose a proteolytic activation model in human skin and confirm differential inhibition of kallikrein (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. They show that these bioactive fragments inhibit KLK-mediated proteolysis of desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling target serine proteinase (SP) activity. PMID:21997416

  19. Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

    PubMed Central

    Bressollier, Philippe; Letourneau, François; Urdaci, Maria; Verneuil, Bernard

    1999-01-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K. PMID:10347045

  20. Competitive inhibition of nitric oxide synthase by p-aminobenzamidine, a serine proteinase inhibitor.

    PubMed

    Venturini, G; Menegatti, E; Ascenzi, P

    1997-03-01

    p-Aminobenzamidine competitively inhibits bovine trypsin, human and bovine thrombin, and human plasmin, all of which act on substrates containing preferentially the L-arginyl side chain at their P1 position. Considering the structural and functional similarity between p-aminobenzamidine and the L-arginyl side chain in trypsin-like serine proteinases, we investigated the interaction of p-aminobenzamidine with mouse brain nitric oxide synthase (NOS), which uses L-arginine as the substrate for generating NO and L-citrulline. p-Aminobenzamidine is a competitive NOS inhibitor (Ki = 1.2 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not an NO precursor. Therefore, p-aminobenzamidine affects the NO production and the trypsin-like serine proteinase action. PMID:9125158

  1. Enhanced response of a proteinase K-based conductometric biosensor using nanoparticles.

    PubMed

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Hamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-01-01

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 µs. These results are greater than that found without any nanoparticles (maximum response of 10 µs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained. PMID:25057139

  2. Enhanced Response of a Proteinase K-Based Conductometric Biosensor Using Nanoparticles

    PubMed Central

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Abdelhamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-01-01

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 μs. These results are greater than that found without any nanoparticles (maximum response of 10 μs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained. PMID:25057139

  3. Modified TB rapid test by proteinase K for rapid diagnosis of pleural tuberculosis.

    PubMed

    Yari, Shamsi; Hadizadeh Tasbiti, Alireza; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Fateh, Abolfazl; Yari, Fatemeh; Bahrmand, Ahmadreza

    2016-03-01

    The diagnosis of pleural tuberculosis continues to be a challenge due to the low sensitivity of traditional diagnostic methods. Better and more rapid tests are needed for diagnosis of pleural TB. In this study, pleural fluids were tested with rapid test to determine Mycobacterium tuberculosis (MTB antigen). Affinity chromatography was used to purify specific polyclonal antibodies against MTB antigen. Pleural samples after decontamination were treated with proteinase K. Rapid test for pleural fluids was prepared by specific antibody. Rapid test was performed on 85 pleural fluid patients. The patients had a mean age of 46.55 ± 15.96 years and 38 were men. The performance of rapid test, using proteinase K, was found to be the most impressive: sensitivity 93%, specificity 94%, PPV 90%, and NPV 96% compared with adenosine deaminase test (ADA), PCR, smear, and culture. The present study did demonstrate that modified TB rapid test can substantially improve the diagnosis of extrapulmonary TB. PMID:26693840

  4. Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

    PubMed Central

    D'Castro, Laura; Wenborn, Adam; Gros, Nathalie; Joiner, Susan; Cronier, Sabrina; Collinge, John; Wadsworth, Jonathan D. F.

    2010-01-01

    Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC. PMID:21187933

  5. The procollagen N-proteinases ADAMTS2, 3 and 14 in pathophysiology.

    PubMed

    Bekhouche, Mourad; Colige, Alain

    2015-01-01

    Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing. PMID:25863161

  6. Programs for high-speed Fourier, Mellin and Fourier-Bessel transforms

    NASA Technical Reports Server (NTRS)

    Ikhabisimov, D. K.; Debabov, A. S.; Kolosov, B. I.; Usikov, D. A.

    1979-01-01

    Several FORTRAN program modules for performing one-dimensional and two-dimensional discrete Fourier transforms, Mellin, and Fourier-Bessel transforms are described along with programs that realize the algebra of high speed Fourier transforms on a computer. The programs can perform numerical harmonic analysis of functions, synthesize complex optical filters on a computer, and model holographic image processing methods.

  7. The fractional Fourier transform and applications

    NASA Technical Reports Server (NTRS)

    Bailey, David H.; Swarztrauber, Paul N.

    1991-01-01

    This paper describes the 'fractional Fourier transform', which admits computation by an algorithm that has complexity proportional to the fast Fourier transform algorithm. Whereas the discrete Fourier transform (DFT) is based on integral roots of unity e exp -2(pi)i/n, the fractional Fourier transform is based on fractional roots of unity e exp -2(pi)i(alpha), where alpha is arbitrary. The fractional Fourier transform and the corresponding fast algorithm are useful for such applications as computing DFTs of sequences with prime lengths, computing DFTs of sparse sequences, analyzing sequences with noninteger periodicities, performing high-resolution trigonometric interpolation, detecting lines in noisy images, and detecting signals with linearly drifting frequencies. In many cases, the resulting algorithms are faster by arbitrarily large factors than conventional techniques.

  8. Protection against ionising radiation and synergism with thiols by zinc aspartate.

    PubMed

    Floersheim, G L; Floersheim, P

    1986-06-01

    Pre-treatment with zinc aspartate protected mice against the lethal effects of radiation and raised the LD50 from 8 Gy to 12.2 Gy. Zinc chloride and zinc sulphate were clearly less active. The radioprotective effect of zinc aspartate was equivalent to cysteamine and slightly inferior to S,2-aminoethylisothiourea (AET). Zinc aspartate displayed a similar therapeutic index to the thiols but could be applied at an earlier time before irradiation. Synergistic effects occurred with the combined administration of zinc aspartate and thiols. By giving zinc aspartate with cysteamine, the LD50 was increased to 13.25 Gy and, by combining it in the optimal protocol with AET, to 17.3 Gy. The radioprotection by zinc and its synergism with thiols is explained by the stabilisation of thiols through the formation of zinc complexes. PMID:3518853

  9. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung

    SciTech Connect

    Pickrell, J.A.; Gregory, R.E.; Cole, D.J.; Hahn, F.F.; Henderson, R.F.

    1987-04-01

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a /sup 14/C-globin substrate. The 48-hr exposures to O/sub 3/ at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O/sub 3/ resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O/sub 3/, which correlated with inflammatory cells noted histologically. At 1.5 ppm O/sub 3/, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O/sub 3/ exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema.

  10. Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi.

    PubMed

    Bontempi, E; Cazzulo, J J

    1990-08-01

    The major cysteine proteinase (cruzipain) from Trypanosoma cruzi was able to digest human IgG, as shown by polyacrylamide gel electrophoresis in the presence of SDS, and by gel filtration on a Superose 12 column, in a FPLC system. The Fab fragment of IgG was only slightly degraded, but Fc was extensively hydrolyzed to small peptides. The results suggest that cruzipain might be involved in the defense mechanisms of the parasite against the immune response of the host. PMID:2227369

  11. SARS CoV Main Proteinase: The Monomer-Dimer Equilibrium Dissociation Constant

    SciTech Connect

    Graziano,V.; McGrath, W.; Yang, L.; Mangel, W.

    2006-01-01

    The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.

  12. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung.

    PubMed

    Pickrell, J A; Gregory, R E; Cole, D J; Hahn, F F; Henderson, R F

    1987-04-01

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a 14C-globin substrate. The 48-hr exposures to O3 at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O3 resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O3, which correlated with inflammatory cells noted histologically. At 1.5 ppm O3, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O3 exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema. PMID:3549351

  13. The Geostationary Fourier Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    Key, Richard; Sander, Stanley; Eldering, Annmarie; Miller, Charles; Frankenberg, Christian; Natra, Vijay; Rider, David; Blavier, Jean-Francois; Bekker, Dmitriy; Wu, Yen-Hung

    2012-01-01

    The Geostationary Fourier Transform Spectrometer (GeoFTS) is an imaging spectrometer designed for an earth science mission to measure key atmospheric trace gases and process tracers related to climate change and human activity. The GeoFTS instrument is a half meter cube size instrument designed to operate in geostationary orbit as a secondary "hosted" payload on a commercial geostationary satellite mission. The advantage of GEO is the ability to continuously stare at a region of the earth, enabling frequent sampling to capture the diurnal variability of biogenic fluxes and anthropogenic emissions from city to continental scales. The science goal is to obtain a process-based understanding of the carbon cycle from simultaneous high spatial resolution measurements of carbon dioxide (CO2), methane (CH4), carbon monoxide (CO), and chlorophyll fluorescence (CF) many times per day in the near infrared spectral region to capture their spatial and temporal variations on diurnal, synoptic, seasonal and interannual time scales. The GeoFTS instrument is based on a Michelson interferometer design with a number of advanced features incorporated. Two of the most important advanced features are the focal plane arrays and the optical path difference mechanism. A breadboard GeoFTS instrument has demonstrated functionality for simultaneous measurements in the visible and IR in the laboratory and subsequently in the field at the California Laboratory for Atmospheric Remote Sensing (CLARS) observatory on Mt. Wilson overlooking the Los Angeles basin. A GeoFTS engineering model instrument is being developed which will make simultaneous visible and IR measurements under space flight like environmental conditions (thermal-vacuum at 180 K). This will demonstrate critical instrument capabilities such as optical alignment stability, interferometer modulation efficiency, and high throughput FPA signal processing. This will reduce flight instrument development risk and show that the Geo

  14. The Geostationary Fourier Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    Key, Richard; Sander, Stanley; Eldering, Annmarie; Blavier, Jean-Francois; Bekker, Dmitriy; Manatt, Ken; Rider, David; Wu, Yen-Hung

    2012-01-01

    The Geostationary Fourier Transform Spectrometer (GeoFTS) is an imaging spectrometer designed for a geostationary orbit (GEO) earth science mission to measure key atmospheric trace gases and process tracers related to climate change and human activity. GEO allows GeoFTS to continuously stare at a region of the earth for frequent sampling to capture the variability of biogenic fluxes and anthropogenic emissions from city to continental spatial scales and temporal scales from diurnal, synoptic, seasonal to interannual. The measurement strategy provides a process based understanding of the carbon cycle from contiguous maps of carbon dioxide (CO2), methane (CH4), carbon monoxide (CO), and chlorophyll fluorescence (CF) collected many times per day at high spatial resolution (2.7kmx2.7km at nadir). The CO2/CH4/CO/CF measurement suite in the near infrared spectral region provides the information needed to disentangle natural and anthropogenic contributions to atmospheric carbon concentrations and to minimize uncertainties in the flow of carbon between the atmosphere and surface. The half meter cube size GeoFTS instrument is based on a Michelson interferometer design that uses all high TRL components in a modular configuration to reduce complexity and cost. It is self-contained and as independent of the spacecraft as possible with simple spacecraft interfaces, making it ideal to be a "hosted" payload on a commercial communications satellite mission. The hosted payload approach for measuring the major carbon-containing gases in the atmosphere from the geostationary vantage point will affordably advance the scientific understating of carbon cycle processes and climate change.

  15. In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents.

    PubMed

    Stepek, Gillian; Lowe, Ann E; Buttle, David J; Duce, Ian R; Behnke, Jerzy M

    2007-12-01

    Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock. PMID:18005461

  16. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

    PubMed

    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. PMID:23619703

  17. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

    PubMed Central

    Burnett, T J; Shankweiler, G W; Hageman, J H

    1986-01-01

    Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase. PMID:3079745

  18. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    PubMed Central

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros) of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized. PMID:26999188

  19. Phage display selection of P1 mutants of BPTI directed against five different serine proteinases.

    PubMed

    Kiczak, L; Koscielska, K; Otlewski, J; Czerwinski, M; Dadlez, M

    1999-01-01

    The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of Ka = 3 x 10(11) M(-1). The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln. PMID:10064144

  20. Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins.

    PubMed

    Torrie, J P; Cholette, H; Froehlich, D A; McKellar, R C

    1983-08-01

    An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk. PMID:6413562

  1. Classification of microbial α-amylases for food manufacturing using proteinase digestion

    PubMed Central

    Akiyama, Takumi; Yamazaki, Takeshi; Tada, Atsuko; Ito, Yusai; Otsuki, Noriko; Akiyama, Hiroshi

    2014-01-01

    Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion–HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups. PMID:25473515

  2. The anthelmintic efficacy of natural plant cysteine proteinases against the rat tapeworm Hymenolepis diminuta in vivo.

    PubMed

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2016-05-01

    Hymenolepis diminuta is a natural parasite of the common brown rat Rattus norvegicus, and provides a convenient model system for the assessment of the anthelmintic activity of novel drugs against cestodes. The experiments described in this paper indicate that treatment of rats infected with H. diminuta with a supernatant extract of papaya latex, containing a mixture of four cysteine proteinases, was moderately efficacious, resulting in a significant, but relatively small, reduction in worm burden and biomass. However, faecal egg output was not affected by treatment. In our experiments these effects were only partially dose-dependent, although specific inhibition by E-64 confirmed the role of cysteine proteinases as the active principles in papaya latex affecting worm growth but not statistically reducing worm burden. Data collected for a further 7 days after treatment indicated that the effects of papaya latex supernatant on worm loss and on worm growth were not enhanced. Our findings provide a starting point for further refinement in formulation and delivery, or assessment of alternative natural plant-derived cysteine proteinases in efforts to develop these naturally occurring enzymes into broad-spectrum anthelmintics, with efficacy against cestodes as well as nematodes. PMID:25761568

  3. Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Zubenko, George S.; Jones, Elizabeth W.

    1981-01-01

    During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation. PMID:7021321

  4. Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins.

    PubMed

    Berge, A; Björck, L

    1995-04-28

    Streptococcus pyogenes are important pathogenic bacteria which produce an extracellular cysteine proteinase contributing to their virulence and pathogenicity. S. pyogenes also express surface molecules, M proteins, that are major virulence determinants due to their antiphagocytic property. In the present work live S. pyogenes bacteria of the M1 serotype were incubated with purified cysteine proteinase. Several peptides were solubilized, and analysis of their protein-binding properties and amino acid sequences revealed two internal fibrinogen-binding fragments of M1 protein (17 and 21 kDa, respectively), and a 36-kDa IgG-binding NH2-terminal fragment of protein H, an IgGFc-binding surface molecule. M protein also plays a role in streptococcal adherence, and removal of this and other surface proteins could promote bacterial dissemination, whereas the generation of soluble complexes between immunoglobulins and immunoglobulin-binding streptococcal surface proteins could be an etiological factor in the development of glomerulonephritis and rheumatic fever. Thus, in these serious complications to S. pyogenes infections immune complexes are found in affected organs. The cysteine proteinase also solubilized a 116-kDa internal fragment of C5a peptidase, another streptococcal surface protein. Activation of the complement system generates C5a, a peptide stimulating leukocyte chemotaxis. C5a-mediated granulocyte migration was blocked by the 116-kDa fragment. This mechanism, by which phagocytes could be prevented from reaching the site of infection, may also contribute to the pathogenicity and virulence of S. pyogenes. PMID:7730368

  5. Snake venoms. The amino acid sequences of two proteinase inhibitor homologues from Dendroaspis angusticeps venom.

    PubMed

    Joubert, F J; Taljaard, N

    1980-05-01

    Toxins C13S1C3 and C13S2C3 from D. angusticeps venom were purified by gel filtration and ion exchange chromatography. Whereas C13S1C3 contains 57 amino acids, C13S2C3 contains 59 but each include six half-cystine residues. The complete primary structure of the low toxicity proteins have been elucidated. The sequences and the invariant residues of toxins C13S1C3 and C13S2C3 from D. angusticeps venom resemble, respectively, those of the proteinase inhibitor homologues K and I from D. polylepis polylepis venom and they are also homologous to the active proteinase inhibitors from various sources. In C13S1C3 and K the active site lysyl residue of active bovine pancreatic proteinase inhibitor is conserved but the site residue alanine, is replaced by lysine. In C13S2C3 and I the active site residue is replaced by tyrosine. PMID:7429422

  6. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells.

    PubMed

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3C(pro)s) of picornaviruses share similar spatial structures and it is becoming apparent that 3C(pro) plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3C(pro) are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3C(pro) can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3C(pro) and these essential factors, 3C(pro) is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3C(pro) are ongoing and a better understanding of the roles played by 3C(pro) may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3C(pro) is summarized. PMID:26999188

  7. [Characteristics of proteinase digestive function in invertebrates--inhabitants of cold seas].

    PubMed

    Mukhin, V A; Smirnova, E B; Novikov, V Iu

    2007-01-01

    Digestive proteinases of various taxa of invertebrates of the Northern seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Grossaster papposus. The presence of two proteolytic activity peaks in the acid (pH 2.5-3.5) and low alkaline zones (pH 7.5-8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH zones is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the low alkaline zone, whereas acid proteolysis has intracellular lysosomal localization. PMID:18038635

  8. Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

    PubMed Central

    Horwitz, Marshall S.; Jenne, Dieter E.; Gauthier, Francis

    2010-01-01

    Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies. PMID:21079042

  9. Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

    PubMed

    Punzo, Daniela; Errico, Francesco; Cristino, Luigia; Sacchi, Silvia; Keller, Simona; Belardo, Carmela; Luongo, Livio; Nuzzo, Tommaso; Imperatore, Roberta; Florio, Ermanno; De Novellis, Vito; Affinito, Ornella; Migliarini, Sara; Maddaloni, Giacomo; Sisalli, Maria Josè; Pasqualetti, Massimo; Pollegioni, Loredano; Maione, Sabatino; Chiariotti, Lorenzo; Usiello, Alessandro

    2016-03-01

    The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. PMID:26961959

  10. An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

    PubMed

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter W; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M

    2015-07-30

    The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  11. Flap Dynamics in Aspartic Proteases: A Computational Perspective.

    PubMed

    Mahanti, Mukul; Bhakat, Soumendranath; Nilsson, Ulf J; Söderhjelm, Pär

    2016-08-01

    Recent advances in biochemistry and drug design have placed proteases as one of the critical target groups for developing novel small-molecule inhibitors. Among all proteases, aspartic proteases have gained significant attention due to their role in HIV/AIDS, malaria, Alzheimer's disease, etc. The binding cleft is covered by one or two β-hairpins (flaps) which need to be opened before a ligand can bind. After binding, the flaps close to retain the ligand in the active site. Development of computational tools has improved our understanding of flap dynamics and its role in ligand recognition. In the past decade, several computational approaches, for example molecular dynamics (MD) simulations, coarse-grained simulations, replica-exchange molecular dynamics (REMD) and metadynamics, have been used to understand flap dynamics and conformational motions associated with flap movements. This review is intended to summarize the computational progress towards understanding the flap dynamics of proteases and to be a reference for future studies in this field. PMID:26872937

  12. Supermacroporous chemically cross-linked poly(aspartic acid) hydrogels.

    PubMed

    Gyarmati, Benjámin; Mészár, E Zsuzsanna; Kiss, Lóránd; Deli, Mária A; László, Krisztina; Szilágyi, András

    2015-08-01

    Chemically cross-linked poly(aspartic acid) (PASP) gels were prepared by a solid-liquid phase separation technique, cryogelation, to achieve a supermacroporous interconnected pore structure. The precursor polymer of PASP, polysuccinimide (PSI) was cross-linked below the freezing point of the solvent and the forming crystals acted as templates for the pores. Dimethyl sulfoxide was chosen as solvent instead of the more commonly used water. Thus larger temperatures could be utilized for the preparation and the drawback of increase in specific volume of water upon freezing could be eliminated. The morphology of the hydrogels was characterized by scanning electron microscopy and interconnectivity of the pores was proven by the small flow resistance of the gels. Compression tests also confirmed the interconnected porous structure and the complete re-swelling and shape recovery of the supermacroporous PASP hydrogels. The prepared hydrogels are of interest for several biomedical applications as scaffolding materials because of their cytocompatibility, controllable morphology and pH-responsive character. PMID:25922304

  13. Rotational-translational fourier imaging system

    NASA Technical Reports Server (NTRS)

    Campbell, Jonathan W. (Inventor)

    2004-01-01

    This invention has the ability to create Fourier-based images with only two grid pairs. The two grid pairs are manipulated in a manner that allows (1) a first grid pair to provide multiple real components of the Fourier-based image and (2) a second grid pair to provide multiple imaginary components of the Fourier-based image. The novelty of this invention resides in the use of only two grid pairs to provide the same imaging information that has been traditionally collected with multiple grid pairs.

  14. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    PubMed

    Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

    2013-09-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone. PMID:23859879

  15. The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

    PubMed Central

    Faehnle, Christopher R.; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E.

    2006-01-01

    The activation of the β-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate l-­aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups. PMID:17012784

  16. FOURIER TRANSFORM INFRARED SPECTROMETRY OF AMBIENT AEROSOLS

    EPA Science Inventory

    Fourier transform infrared (FTIR) spectrometry has been evaluated as a method for determining the concentration of selected species present in ambient aerosols collected on Teflon filters. The filters are analyzed by transmission measurements after collection of the fine fraction...

  17. Plasma Spectrochemistry with a Fourier Transform Spectrometer.

    NASA Astrophysics Data System (ADS)

    Manning, Thomas Joseph John

    1990-01-01

    This dissertation can be interpreted as being two-dimensional. The first dimension uses the Los Alamos Fourier Transform Spectrometer to uncover various physical aspects of a Inductively Coupled Plasma. The limits of wavenumber accuracy and resolution are pushed to measure line shifts and line profiles in an Inductively Coupled Argon Plasma. This is new physical information that the plasma spectroscopy community has been seeking for several years. Other plasma spectroscopy carried out includes line profile studies, plasma diagnostics, and exact identification of diatomic molecular spectra. The second aspect of the dissertation involves studies of light sources for Fourier Transform Spectroscopy. Sources developed use an inductively coupled plasma (ICP) power supply. New sources (neon ICP, closed cell ICP, and helium ICP) were developed and new methods to enhance the performance and understand a Fourier Transform Spectrometer were studied including a novel optical filter, a spectrum analyzer to study noises, and a standard to calibrate and evaluate a Fourier Transform Spectrometer.

  18. Electronically-Scanned Fourier-Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    Breckinridge, J. B.; Ocallaghan, F. G.

    1984-01-01

    Instrument efficient, lightweight, and stable. Fourier-transform spectrometer configuration uses electronic, instead of mechanical, scanning. Configuration insensitive to vibration-induced sampling errors introduced into mechanically scanned systems.

  19. Didactic toy for teaching the Fourier principle

    NASA Astrophysics Data System (ADS)

    Medina-Villanueva, Miguel; Medina-Tamez, Victor; Medina-Tamez, Javier; Garcia-Mederez, Adrian

    2002-11-01

    The mathematical tool of Fourier analysis is used in many areas like vibrations, communications, optics, electronics, etc. The understanding of this subject sometimes causes frustration in students. The main purpose of this presentation is to propose a didactic toy that calculates the harmonic magnitudes through the discrete values of analog periodic signals. This device shows the rotative vectors in a physical way that makes the principle of Fourier understandable.

  20. Insulin degludec aspart: One-year real world experience

    PubMed Central

    Kalra, Sanjay; Baruah, Manash P.

    2016-01-01

    Background: This retrospective analysis describes the use of insulin degludec aspart (IDegAsp) in India. Material and Methods: All subjects who had received IDegAsp for 52 weeks at two endocrine centers were included in this study. Results: Forty-eight subjects (40 men), with mean age of 54.33 ± 9.63 years and mean duration of diabetes of 6.33 ± 2.96 years, started IDegAsp as insulin of initiation (16), as an intensification regime (4), as de-escalation from basal-bolus therapy (16), or as switch from premixed insulin (12). The dose of IDegAsp fell from 43.17 ± 21.18 U/day or 0.56 ± 0.23 U/kg to 37.75 ± 17.13U/day (0.51 ± 0.12 U/kg) at 24 weeks and 41.41 ± 15.33 U/day (0.56 ± 0.17 U/kg) at 52 weeks. Hemoglobin A1c (HbA1c), which was 9.52 ± 1.27% at the start of therapy, fell to 7.51 ± 0.46% at 26 weeks and to 7.48 ± 0.40% at 52 weeks. Fasting plasma glucose fell from 154.08 ± 33.30 mg% to 108.58 ± 22.26 mg% at 26 weeks and 102.17 ± 12.79 mg% at 52 weeks. Of the 48 subjects, 39 (81.25%) achieved a target of HbA1c <7.0% at both 26 and 52 weeks. No episode of hypoglycemia was reported in the 4 weeks preceding the analysis. Conclusion: This communication highlights the efficacy, safety, and tolerability, while providing insight into the usage patterns of IDegAsp. PMID:27186556

  1. Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates.

    PubMed

    Asbóth, B; Stokum, E; Khan, I U; Polgár, L

    1985-01-29

    To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases. PMID:3888259

  2. Pathobiochemical significance of granulocyte elastase complexed with proteinase inhibitors: effect on glycosaminoglycan metabolism in cultured synovial cells.

    PubMed

    Kleesiek, K; van de Leur, E; Reinards, R; Greiling, H

    1987-03-01

    Interactions between elastase inhibitor complexes and synovial cells are of special interest, since, in chronic joint diseases, granulocytes release large amounts of elastase into the synovial fluid and connective tissue, where the proteinase is bound to alpha 1-proteinase inhibitor and alpha 2-macroglobulin. To study the effect of elastase-alpha 2-macroglobulin and elastase-alpha 1-proteinase inhibitor complexes on the glycosaminoglycan metabolism of cultured synovial cells, we determined the distribution of [3H]glucosamine-labelled hyaluronate, which represents the main synthesized glycosaminoglycan, and of 35SO4(2-)-labelled chondroitin sulphate into the intracellular, pericellular and extracellular compartments of the cell culture. Exposure of the synovial cells to elastase-alpha 2-macroglobulin complexes leads to an enhanced synthesis and secretion of hyaluronate, and chondroitin sulphate, and also induces a rise of the fibronectin concentration in the medium. Analogous but less pronounced effects are observed in the presence of elastase-alpha 1-proteinase inhibitor complexes. Native uncomplexed elastase, however, causes no significant changes in hyaluronate metabolism. An increase of prostaglandin E2 in the culture medium during incubation with elastase inhibitor complexes occurs in parallel to the stimulatory effect on glycosaminoglycan metabolism. Our results demonstrate that elastase, whose enzymic activity is inactivated by the formation of complexes with alpha 1-proteinase inhibitor or alpha 2-macroglobulin, nevertheless acts as an inflammatory mediator, which in vitro induces metabolic changes closely resembling the in vivo findings in inflammatory joint diseases. PMID:2439645

  3. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

    SciTech Connect

    Steinberger, Jutta; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.

  4. Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae).

    PubMed

    Fabrick, J; Behnke, C; Czapla, T; Bala, K; Rao, A G; Kramer, K J; Reeck, G R

    2002-04-01

    The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species. PMID:11886775

  5. Adsorption of L-glutamic acid and L-aspartic acid to γ-Al2O3

    NASA Astrophysics Data System (ADS)

    Greiner, Edward; Kumar, Kartik; Sumit, Madhuresh; Giuffre, Anthony; Zhao, Weilong; Pedersen, Joel; Sahai, Nita

    2014-05-01

    The interactions of amino acids with mineral surfaces have potential relevance for processes ranging from pre-biotic chemistry to biomineralization to protein adsorption on biomedical implants in vivo. Here, we report the results of experiments investigating the adsorption of L-glutamic (Glu) and L-aspartic (Asp) acids to γ-Al2O3. We examined the extent of Glu and Asp coverage as a function of pH and solution concentration (pH edges and isotherms) in solution-depletion experiments and used in situ Attenuated Total Refkectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to estimate the molecular conformations of the adsorbed molecules. Glu and Asp exhibited similar adsorption behavior on γ-Al2O3 with respect to pH and solution concentration. In general, adsorption decreased as pH increased. At low and high amino acid concentrations, the isotherms exhibited two apparent saturation coverages, which could be interpreted as 1:4 or 1:2 ratios of adsorbed molecule/surface Al sites. Tetradentate tetranuclear and bidentate binuclear species were the dominant conformations inferred independently from FTIR spectra. In these conformations, both carboxylate groups are involved in bonding to either four or to two Al surface atoms, through direct covalent bonds or via H-bonds. An outer sphere species, in which one carboxylate group interacts with a surface Al atom, could not be ruled out based on the FTIR spectra.

  6. Aspartic acid-based modified PLGA-PEG nanoparticles for bone targeting: in vitro and in vivo evaluation.

    PubMed

    Fu, Yin-Chih; Fu, Tzu-Fun; Wang, Hung-Jen; Lin, Che-Wei; Lee, Gang-Hui; Wu, Shun-Cheng; Wang, Chih-Kuang

    2014-11-01

    Nanoparticles (NP) that target bone tissue were developed using PLGA-PEG (poly(lactic-co-glycolic acid)-polyethylene glycol) diblock copolymers and bone-targeting moieties based on aspartic acid, (Asp)(n(1,3)). These NP are expected to enable the transport of hydrophobic drugs. The molecular structures were examined by (1)H NMR or identified using mass spectrometry and Fourier transform infrared (FT-IR) spectra. The NP were prepared using the water miscible solvent displacement method, and their size characteristics were evaluated using transmission electron microscopy (TEM) and dynamic light scattering. The bone targeting potential of the NP was evaluated in vitro using hydroxyapatite affinity assays and in vivo using fluorescent imaging in zebrafish and rats. It was confirmed that the average particle size of the NP was <200 nm and that the dendritic Asp3 moiety of the PLGA-PEG-Asp3 NP exhibited the best apatite mineral binding ability. Preliminary findings in vivo bone affinity assays in zebrafish and rats indicated that the PLGA-PEG-ASP3 NP may display increased bone-targeting efficiency compared with other PLGA-PEG-based NP that lack a dendritic Asp3 moiety. These NP may act as a delivery system for hydrophobic drugs, warranting further evaluation of the treatment of bone disease. PMID:25050775

  7. Synthesis and Properties of pH-, Thermo-, and Salt-Sensitive Modified Poly(aspartic acid)/Poly(vinyl alcohol) IPN Hydrogel and Its Drug Controlled Release.

    PubMed

    Lu, Jingqiong; Li, Yinhui; Hu, Deng; Chen, Xiaoling; Liu, Yongmei; Wang, Liping; Zhao, Yansheng

    2015-01-01

    Modified poly(aspartic acid)/poly(vinyl alcohol) interpenetrating polymer network (KPAsp/PVA IPN) hydrogel for drug controlled release was synthesized by a simple one-step method in aqueous system using poly(aspartic acid) grafting 3-aminopropyltriethoxysilane (KH-550) and poly(vinyl alcohol) (PVA) as materials. The hydrogel surface morphology and composition were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The thermal stability was analyzed by thermogravimetric analysis (TGA). The swelling properties and pH, temperature, and salt sensitivities of KPAsp, KPAsp/PVA semi-interpenetrating polymer network (semi-IPN), and KPAsp/PVA IPN hydrogels were also investigated. All of the three hydrogels showed ampholytic pH-responsive properties, and swelling behavior was also extremely sensitive to the temperature, ionic strength, and cationic species. Finally, the drug controlled release properties of the three hydrogels were evaluated and results indicated that three hydrogels could control drug release by external surroundings stimuli. The drug controlled release properties of KPAsp/PVA IPN hydrogel are the most outstanding, and the correlative measured release profiles of salicylic acid at 37°C were 32.6 wt% at pH = 1.2 (simulated gastric fluid) and 62.5 wt% at pH = 7.4 (simulated intestinal fluid), respectively. These results indicated that KPAsp/PVA IPN hydrogels are a promising carrier system for controlled drug delivery. PMID:26351630

  8. Synthesis and Properties of pH-, Thermo-, and Salt-Sensitive Modified Poly(aspartic acid)/Poly(vinyl alcohol) IPN Hydrogel and Its Drug Controlled Release

    PubMed Central

    Lu, Jingqiong; Li, Yinhui; Hu, Deng; Chen, Xiaoling; Liu, Yongmei; Wang, Liping; Zhao, Yansheng

    2015-01-01

    Modified poly(aspartic acid)/poly(vinyl alcohol) interpenetrating polymer network (KPAsp/PVA IPN) hydrogel for drug controlled release was synthesized by a simple one-step method in aqueous system using poly(aspartic acid) grafting 3-aminopropyltriethoxysilane (KH-550) and poly(vinyl alcohol) (PVA) as materials. The hydrogel surface morphology and composition were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The thermal stability was analyzed by thermogravimetric analysis (TGA). The swelling properties and pH, temperature, and salt sensitivities of KPAsp, KPAsp/PVA semi-interpenetrating polymer network (semi-IPN), and KPAsp/PVA IPN hydrogels were also investigated. All of the three hydrogels showed ampholytic pH-responsive properties, and swelling behavior was also extremely sensitive to the temperature, ionic strength, and cationic species. Finally, the drug controlled release properties of the three hydrogels were evaluated and results indicated that three hydrogels could control drug release by external surroundings stimuli. The drug controlled release properties of KPAsp/PVA IPN hydrogel are the most outstanding, and the correlative measured release profiles of salicylic acid at 37°C were 32.6 wt% at pH = 1.2 (simulated gastric fluid) and 62.5 wt% at pH = 7.4 (simulated intestinal fluid), respectively. These results indicated that KPAsp/PVA IPN hydrogels are a promising carrier system for controlled drug delivery. PMID:26351630

  9. N-Methyl-D-Aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity

    EPA Science Inventory

    N-Methyl-D-aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity Glufosinate (GLF) at high levels in mammals causes convulsions through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamate...

  10. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells

    PubMed Central

    Sullivan, Lucas B.; Gui, Dan Y.; Hosios, Aaron M.; Bush, Lauren N.; Freinkman, Elizaveta; Vander Heiden, Matthew G.

    2015-01-01

    Summary Mitochondrial respiration is important for cell proliferation, however the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. PMID:26232225

  11. Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

    PubMed

    Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

    2015-07-30

    Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. PMID:26232225

  12. Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

    PubMed

    El-Ahwany, Eman; Rabia, Ibrahim; Nagy, Faten; Zoheiry, Mona; Diab, Tarek; Zada, Suher

    2012-03-01

    Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships. PMID:22451733

  13. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    PubMed

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small

  14. Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells

    SciTech Connect

    Small-Howard, Andrea; Turner, Helen . E-mail: hturner@queens.org

    2005-04-15

    Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

  15. Analysis of a Delivery Device Conversion for Insulin Aspart: Potential Clinical Impact in Veterans.

    PubMed

    Moorman Spangler, Caitlin M; Greck, Beth D; Killian, Jancy H

    2016-04-01

    In Brief Insulin therapies using a wide variety of delivery devices are available to accommodate individual patients' needs. In this study of veterans with diabetes, converting from insulin aspart delivered with vials and syringes to insulin aspart delivered via a pen device resulted in no significant change in A1C. Although insulin pen delivery devices offer benefits, providers should thoroughly consider all potential reasons for uncontrolled diabetes before modifying a patient's insulin delivery method. PMID:27092019

  16. Aspartic Peptidases of Human Pathogenic Trypanosomatids: Perspectives and Trends for Chemotherapy

    PubMed Central

    Santos, L.O.; Garcia-Gomes, A.S.; Catanho, M.; Sodré, C.L.; Santos, A.L.S.; Branquinha, M.H.; d’Avila-Levy, C.M.

    2013-01-01

    Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas’ disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in

  17. Insulin Aspart in the Management of Diabetes Mellitus: 15 Years of Clinical Experience.

    PubMed

    Hermansen, Kjeld; Bohl, Mette; Schioldan, Anne Grethe

    2016-01-01

    Limiting excessive postprandial glucose excursions is an important component of good overall glycemic control in diabetes mellitus. Pharmacokinetic studies have shown that insulin aspart, which is structurally identical to regular human insulin except for the replacement of a single proline amino acid with an aspartic acid residue, has a more physiologic time-action profile (i.e., reaches a higher peak and reaches that peak sooner) than regular human insulin. As expected with this improved pharmacokinetic profile, insulin aspart demonstrates a greater glucose-lowering effect compared with regular human insulin. Numerous randomized controlled trials and a meta-analysis have also demonstrated improved postprandial control with insulin aspart compared with regular human insulin in patients with type 1 or type 2 diabetes, as well as efficacy and safety in children, pregnant patients, hospitalized patients, and patients using continuous subcutaneous insulin infusion. Studies have demonstrated that step-wise addition of insulin aspart is a viable intensification option for patients with type 2 diabetes failing on basal insulin. Insulin aspart has shown a good safety profile, with no evidence of increased receptor binding, mitogenicity, stimulation of anti-insulin antibodies, or hypoglycemia compared with regular human insulin. In one meta-analysis, there was evidence of a lower rate of nocturnal hypoglycemia compared with regular human insulin and, in a trial that specifically included patients with a history of recurrent hypoglycemia, a significantly lower rate of severe hypoglycemic episodes. The next generation of insulin aspart (faster-acting insulin aspart) is being developed with a view to further improving on these pharmacokinetic/pharmacodynamic properties. PMID:26607485

  18. Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

    PubMed Central

    Ford, G C; Eichele, G; Jansonius, J N

    1980-01-01

    X-ray diffraction studies to 2.8-A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous alpha 2 dimer (Mr = 2 x 45,000). The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them. PMID:6930651

  19. Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species.

    PubMed

    Farnham, M W; Griffith, S M; Miller, S S; Vance, C P

    1990-12-01

    Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. PMID:16667896

  20. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    SciTech Connect

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  1. Biochemical features, molecular biology and clinical relevance of the human 15-domain serine proteinase inhibitor LEKTI.

    PubMed

    Walden, Michael; Kreutzmann, Peter; Drögemüller, Katrin; John, Harald; Forssmann, Wolf-Georg; Hans-Jürgen, Mägert

    2002-01-01

    Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsin-inhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable. PMID:12437098

  2. Effect of nonsteroidal antiinflammatory drugs on the neutrophil promoted inactivation of alpha-1-proteinase inhibitor.

    PubMed

    Dallegri, F; Ottonello, L; Dapino, P; Sacchetti, C

    1992-03-01

    We investigated the effect of some nonsteroidal antiinflammatory drugs (aspirin, naproxen and nimesulide) on the ability of neutrophils to oxidatively inactivate the alpha-1-proteinase inhibitor (A1PI). Nimesulide prevented the inactivation of A1PI by effectively scavenging the hypochlorous acid released by neutrophils. Aspirin and naproxen were completely ineffective. We suggest that the antiinflammatory effect of nimesulide may be due at least in part to the rescue of A1PI from neutrophil oxidative attack. The rescue of A1PI may in fact alter the elastase-A1PI balance in favor of the inhibitor, with resulting tissue protection. PMID:1578457

  3. Synthetic analogues of chymostatin. Inhibition of chymotrypsin and Streptomyces griseus proteinase A.

    PubMed Central

    Tomkinson, N P; Galpin, I J; Beynon, R J

    1992-01-01

    A series of analogues of chymostatin, including Z-Arg-Leu-Phe-aldehyde (Z-Arg-Leu-Phe-H), have been synthesized. Analysis of the inhibitory potential of these analogues permits identification of residues and interactions that are important for inhibitory activity. Moreover, the structure-function relationship for Z-Arg-Leu-Phe-H and chymostatin inhibition of chymotrypsin and Streptomyces griseus proteinase A (SGPA) was probed further with the aid of molecular mechanics. This analysis identified interactions that provide an explanation for the enhanced activity of the natural product, chymostatin, over the synthetic analogues in the inhibition of chymotrypsin but not SGPA. PMID:1530579

  4. Lowered circulating aspartate is a metabolic feature of human breast cancer

    PubMed Central

    Xie, Guoxiang; Zhou, Bingsen; Zhao, Aihua; Qiu, Yunping; Zhao, Xueqing; Garmire, Lana; Shvetsov, Yurii B.; Yu, Herbert; Yen, Yun; Jia, Wei

    2015-01-01

    Distinct metabolic transformation is essential for cancer cells to sustain a high rate of proliferation and resist cell death signals. Such a metabolic transformation results in unique cellular metabolic phenotypes that are often reflected by distinct metabolite signatures in tumor tissues as well as circulating blood. Using a metabolomics platform, we find that breast cancer is associated with significantly (p = 6.27E-13) lowered plasma aspartate levels in a training group comprising 35 breast cancer patients and 35 controls. The result was validated with 103 plasma samples and 183 serum samples of two groups of primary breast cancer patients. Such a lowered aspartate level is specific to breast cancer as it has shown 0% sensitivity in serum from gastric (n = 114) and colorectal (n = 101) cancer patients. There was a significantly higher level of aspartate in breast cancer tissues (n = 20) than in adjacent non-tumor tissues, and in MCF-7 breast cancer cell line than in MCF-10A cell lines, suggesting that the depleted level of aspartate in blood of breast cancer patients is due to increased tumor aspartate utilization. Together, these findings suggest that lowed circulating aspartate is a key metabolic feature of human breast cancer. PMID:26452258

  5. A mutant of Arabidopsis thaliana (L.) Heynh. with modified control of aspartate kinase by threonine.

    PubMed

    Heremans, B; Jacobs, M

    1997-04-01

    Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grown on 2 mM lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 x RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1 and ak2) were found to be unlinked. PMID:9241437

  6. Fibrinogen degradation by two neutral granulocyte proteinases. Influence of calcium on the generation of fibrinogen degradation products with anticlotting properties.

    PubMed

    Bingenhkeimer, C; Gramse, M; Egbring, R; Havemann, K

    1981-07-01

    Degradation of human fibrinogen by elastase-like proteinase, chymotrypsin-like proteinase and plasmin, was done in the presence and absence of calcium ions, respectively. The resulting fibrinogen degradation products were tested for their coagulant and anti-coagulant properties. The results show that 1. fibrinogenolysis is delayed in the presence of calcium ions. Higher enzyme concentrations are required to get unclottable split products when calcium ions are present. 2. The fibrinogen fragments obtained in the presence of calcium are different in their molecular weights and anticoagulant activities compared to those obtained in the absence of calcium ions. This effect of calcium is most striking during fibrinogen cleavage by chymotrypsin-like proteinase. Elastase and plasmin-induced fibrinogenolysis was substantially influenced by calcium only at a late degradation stage. PMID:6456216

  7. Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream (Pagrus major).

    PubMed

    Wu, Guo-Ping; Chen, Su-Hua; Liu, Guang-Ming; Yoshida, Asami; Zhang, Ling-Jing; Su, Wen-Jin; Cao, Min-Jie

    2010-03-01

    A collagenolytic serine proteinase (CSP) was purified from red sea bream (Pagrus major) skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Phenyl Sepharose and Hydroxyapatite. The molecular mass of CSP was approximately 85 kDa as estimated by SDS-PAGE and gel filtration. Optimum temperature and pH of CSP were 40 degrees C and 8.0, respectively. CSP was specifically inhibited by serine proteinase inhibitors, while inhibitors to other type proteinases did not show much inhibitory effects. The K(m) and k(cat) values of CSP for Boc-Leu-Lys-Arg-MCA were 3.58 microM and 0.13 s(-1) at 37 degrees C, respectively. Furthermore, CSP hydrolyzed gelatin and native type I collagen effectively though its degradation on myosin heavy chain (MHC) was not significant, suggesting its involvement in the texture tenderization of fish muscle during the post-mortem stage. PMID:19945542

  8. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds.

    PubMed

    Patil, Dipak N; Chaudhry, Anshul; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra

    2009-07-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS-PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 A. Diffraction data were collected to a resolution of 2.7 A. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%. PMID:19574654

  9. 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 ...

  10. Potential Use of Proteinase Inhibitors, Avidin, and Other Bio-reagents for Synergizing Bt Performance and Delaying Resistance Development to Bt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After being ingested by target insects, the insecticidal proteins from Bacillus thuringiensis (Bt) need to go through a proteolytic process by insect midgut proteinases to become activated. At the same time, Bt can be hydrolyzed and degraded by midgut proteinases to become non-toxic to target insect...

  11. A Cooperative Escherichia coli Aspartate Transcarbamoylase without Regulatory Subunits

    SciTech Connect

    Mendes, K.; Kantrowitz, E

    2010-01-01

    Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c{sub 6}r{sub 6}. Dissociation of the native holoenzyme produces catalytically active trimers, c{sub 3}, and nucleotide-binding regulatory dimers, r{sub 2}. By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c{sub 6}, was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c{sub 6} species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c3. The c{sub 6} ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 {angstrom} resolution. The structure of c{sub 6} ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c{sub 6} enzyme, enhancing its activity over the wild-type holoenzyme (c{sub 6}r{sub 6}) and c{sub 3}. The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction

  12. L-aspartic acid transport by cat erythrocytes

    SciTech Connect

    Chen, C.W.; Preston, R.L.

    1986-03-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of /sup 3/H-L-asp (typically 2..mu..M) was measured in washed RBCs incubated for 60 s at 37/sup 0/C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl/sub 2/, 15 mM MOPS pH 7.4, 5 mM glucose, and /sup 14/C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000..mu..M, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 ..mu..M and 148.8 +/- 7.2 ..mu..mol 1. cell/sup -1/h/sup -1/ respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4/sup +/M L-asp, 40/sup +/M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues.

  13. Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.

    PubMed Central

    Verchot, J; Carrington, J C

    1995-01-01

    The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification. PMID:7745715

  14. The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

    PubMed Central

    Gobbetti, M; Smacchi, E; Corsetti, A

    1996-01-01

    A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively. PMID:8795211

  15. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

    PubMed

    Yang, Hu; Zhai, Chao; Yu, Xianhong; Li, Zhezhe; Tang, Wei; Liu, Yunyun; Ma, Xiaojian; Zhong, Xing; Li, Guolong; Wu, Di; Ma, Lixin

    2016-06-01

    Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively. PMID:26892536

  16. [Features of clinical course and proteinase inhibitor balance in tears in eye burns of different localization (an experimental study)].

    PubMed

    Chesnokova, N B; Malarpv, P V; Beznos, O V

    2001-01-01

    Chemical burns of the eye of different location (cornea, corneal fragment with adjacent conjunctiva, and limbus) were studied in experiments. Clinical picture and changes in the lacrimal proteinase inhibitor balance were analyzed. Burn disease is less severe and the number of complications is less if a fragment of the cornea with adjacent conjunctiva and a fragment of the limbus are injured than in case of a corneal burn of the same depth and area. Burn of the total limbus area is a severe injury involving essential shifts in the proteinase inhibitor balance, leading to deep organic changes in the cornea and inner structures of the eye, eventuating in its subatrophy. PMID:11510165

  17. Anti-epidermal-cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase.

    PubMed Central

    Farb, R M; Dykes, R; Lazarus, G S

    1978-01-01

    Immunoglobulin from pemphigus patients binds to the surface of mouse epidermal cells in culture. Cells incubated with the pemphigus antibody are easily detached from culture plates whereas cells incubated with serum from normal patients remain on the plate. Pemphigus antibody-mediated cell detachment is blocked by the addition of the proteinase inhibitors soybean trypsin inhibitor and alpha2-macroglobulin to the culture media. Detachable cells are viable, and activation of the complement cascade is not necessary for cell detachment. The anti-cell-surface antibody of pemphigus appears to disrupt adhesion between viable epidermal cells by activation of proteinase. Images PMID:272663

  18. A Fourier approach to cloud motion estimation

    NASA Technical Reports Server (NTRS)

    Arking, A.; Lo, R. C.; Rosenfeld, A.

    1978-01-01

    A Fourier phase-difference technique for cloud motion estimation from pairs of pictures is described, and results obtained using this technique are compared with the results of a Fourier-domain cross-correlation scheme. The phase-difference technique makes use of the phase of the cross-spectral density and allows motion estimates to be made for individual spatial frequencies, which are related to cloud pattern dimensions. When objects being tracked do not change their shape, size, and orientation to more than a limited degree, both techniques are effective. The phase difference technique is relatively sensitive to the presence of mixtures of motions, changes in cloud shape, and edge effects; in these circumstances, the cross-correlation scheme is preferable. It is suggested that the Fourier transform phase difference estimation methods can be applied in problems such as landmark matching.

  19. Fast Fourier Transforms of Piecewise Constant Functions

    NASA Astrophysics Data System (ADS)

    Sorets, Eugene

    1995-02-01

    We present an algorithm for the evaluation of the Fourier transform of piecewise constant functions of two variables. The algorithm overcomes the accuracy problems associated with computing the Fourier transform of discontinuous functions; in fact, its time complexity is O (N2 logN + NP log2 (1/ε) + V log3 (1/ε)), where ε is the accuracy, N is the size of the problem, P is the perimeter of the set of discontinuities, and V is its number of vertices. The algorithm is based on the Lagrange interpolation formula and the Green's theorem, which are used to preprocess the data before applying the fast Fourier transform. It readily generalizes to higher dimensions and to piecewise smooth functions.

  20. Analysis of eccentric photorefraction by Fourier optics

    NASA Astrophysics Data System (ADS)

    Liu, Yong; Chen, Jiabi

    2007-03-01

    Eccentric photorefraction usually is used as early eyesight diagnostic test of infants and small children. Unlike currently approved geometrical optical model of eccentric photorefractometer, the crescent formation and the light-intensity distribution in the pupil image of a myopic eye are analyzed by Fourier optics with the assumption of an isotropic scattering retina. In the case of little circular light source and rectangular slit, the simulation results of different myopic diopters are obtained by geometrical optical theory and Fourier optics respectively. It is found that the simulation results by Fourier optics are similar as those obtained by geometrical optics, and all simulations are almost corresponding to the experimental result. The result demonstrates that the new method presented here is feasible.

  1. Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils.

    PubMed

    Sallenave, J M; Si Tahar, M; Cox, G; Chignard, M; Gauldie, J

    1997-06-01

    Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be up-regulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype. PMID:9201260

  2. Secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome.

    PubMed

    Sallenave, J M; Donnelly, S C; Grant, I S; Robertson, C; Gauldie, J; Haslett, C

    1999-05-01

    Inappropriate release of proteases from inflammatory and stromal cells can lead to destruction of the lung parenchyma. Antiproteinases such as alpha-1-proteinase inhibitor (alpha1-Pi), secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (elafin) control excess production of human neutrophil elastase. In the present study, the concentrations of alpha1-Pi, SLPI and elafin found in bronchoalveolar lavage (BAL) fluid from control subjects, patients at risk of developing acute respiratory distress syndrome (ARDS) and patients with established ARDS were determined. Levels of all three inhibitors were raised in patients compared with normal subjects. SLPI was increased in the group of patients who were at risk of ARDS and went on to develop the condition, compared with the "at-risk" group who did not progress to ARDS (p=0.0083). Alpha1-Pi and elafin levels were similar in these two populations. In patients with established ARDS, both alpha1-Pi and SLPI levels were significantly increased, compared to patients at risk of ARDS who did (p=0.0089) or did not (p=0.0003) progress to ARDS. The finding of increased antiproteinases shortly before the development of acute respiratory distress syndrome provide further evidence for enhanced inflammation prior to clinical disease. PMID:10414400

  3. [Purification and properties of serine proteinases from European catfish Silurus glanis L. pancreas].

    PubMed

    Ulitina, N N; Khabliuk, V V; Proskuriakov, M T

    2005-01-01

    Three trypsin isoforms (designated as T1, T2, and T3), three chymotrypsin isoforms (Kh1, Kh2, and Kh3), and two elastase isoforms (E1 and E2) were isolated from the pancreas of European catfish Silurus glanis L. by salting out with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion exchange chromatography on DEAE cellulose. Isoelectric points of the enzymes, determined by isoelectric focusing, amounted to 4.42 for T1, 5.64 for T2, 6.90 for T3, 4.93 for Khl, 5.23 for Kh2, 6.18 for Kh3, 6.17 for E1, and 8.48 for E2. Molecular weights of proteinases within each group were close and amounted to 30100 Da for trypsins, 39800 Da for chymotrypsins, and 24000 Da for elastases. The enzymes isolated displayed maximal activities at alkaline pH values. Inhibitor analysis demonstrated that all the proteinases isolated from European catfish pancreas belonged to the serine type. PMID:15859457

  4. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

    PubMed

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-07-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  5. [Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa].

    PubMed

    Vakorina, T I; Gladkikh, I N; Monastyrnaia, M M; Kozlovskaia, E P

    2011-01-01

    The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available. PMID:21899045

  6. Candida tropicalis Biofilms: Biomass, Metabolic Activity and Secreted Aspartyl Proteinase Production.

    PubMed

    Negri, Melyssa; Silva, Sónia; Capoci, Isis Regina Grenier; Azeredo, Joana; Henriques, Mariana

    2016-04-01

    According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24-120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation. PMID:26572148

  7. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

    PubMed

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. PMID:26907253

  8. Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis.

    PubMed

    Dekeyser, P M; Buttle, D J; Devreese, B; Van Beeumen, J; Demeester, J; Lauwers, A

    1995-07-10

    The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis. PMID:7625846

  9. Proteinases, their receptors and inflammatory signalling: the Oxford South Parks Road connection*

    PubMed Central

    Hollenberg, M D

    2015-01-01

    In keeping with the aim of the Paton Memorial Lecture to ‘facilitate the historical study of pharmacology’, this overview, which is my distinct honour to write, represents a ‘Janus-like’ personal perspective looking both backwards and forwards at the birth and growth of ‘receptor molecular pharmacology’ with special relevance to inflammatory diseases. The overview begins in the Oxford Department of Pharmacology in the mid-1960s and then goes on to provide a current perspective of signalling by proteinases. Looking backwards, the synopsis describes the fruitful Oxford Pharmacology Department infrastructure that Bill Paton generated in keeping with the blueprint begun by his predecessor, J H Burn. Looking forwards, the overview illustrates the legacy of that environment in generating some of the first receptor ligand-binding data and providing the inspiration and vision for those like me who were training in the department at the same time. With apologies, I mention only in passing a number of individuals who benefitted from the ‘South Parks Road connection’ using myself as one of the ‘outcome study’ examples. It is also by looking forward that I can meet the complementary aim of summarizing the lecture presented at a ‘BPS 2014 Focused Meeting on Cell Signalling’ to provide an overview of the role of proteinases and their signalling mechanisms in the setting of inflammation. PMID:25521749

  10. Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer

    SciTech Connect

    Xu, Ting; Ooi, Amy; Lee, Hooi Chen; Wilmouth, Rupert; Liu, Ding Xiang; Lescar, Julien

    2005-11-01

    An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.

  11. Purification and Developmental Analysis of an Extracellular Proteinase from Young Leaves of Soybean.

    PubMed Central

    Huangpu, J.; Graham, J. S.

    1995-01-01

    A proteinase present in intercellular wash fluids from leaves of Glycine max has been purified 600-fold to electrophoretic homogeneity. The native protein is monomeric with a molecular mass of 60 kD, as estimated by denaturing gel electrophoresis, and has an isoelectric point of 7.7. The enzyme has a pH optimum of 9.5 when assayed with Azocoll as a substrate. The proteolytic activity is inhibited by p-chloromercuribenzoic acid and mercuric chloride and requires the presence of reducing agents. The enzyme activity is refractory to other classical sulfhydryl proteinases. The soybean leaf endoproteinase is present within the extracellular space of young leaves, and a portion is bound to the cell wall. Western blot analysis and activity measurements show that the enzyme is present only during the first 15 d postemergence of the leaf and is therefore under strict developmental control. We suggest that the enzyme may play a critical role in the extracellular milieu during rapid cell growth and leaf expansion. PMID:12228520

  12. Uniquely Regulated Proteinase Inhibitor I Gene in a Wild Tomato Species 1

    PubMed Central

    Wingate, Vincent P. M.; Ryan, Clarence A.

    1991-01-01

    A uniquely regulated proteinase inhibitor I gene was isolated from the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The inhibitor gene is wound-inducible in leaves and is expressed in unripe fruit during development. The gene (λ clone 1) is present on a 15.5 kilobase pair Sal 1-SalI genomic DNA fragment. Southern blot analysis of L. peruvianum genomic DNA shows only one strongly hybridizing DNA fragment to probes derived from λ clone 1. S1 nuclease protection experiments and Northern analyses confirm that this gene is both wound-inducible in leaves and developmentally regulated in young unripe fruit. These observations are supported by comparisons of the 5′-flanking DNA sequences of the L. peruvianum inhibitor I gene with known elicitor responsive cis-acting sequences. The transcriptional regulation of the λ clone 1 inhibitor I gene in leaves of wounded plants and in developing unripe fruit indicates that the gene contains unique complex regulating elements. These elements respond to both environmental and developmental tissue-specific signals that can regulate proteinase inhibitor synthesis to protect the tissues of this wild species of tomato against predators and pathogens. ImagesFigure 4Figure 5Figure 6 PMID:16668426

  13. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

    PubMed Central

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. PMID:26907253

  14. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

    PubMed

    von Pawel-Rammingen, Ulrich; Johansson, Björn P; Björck, Lars

    2002-04-01

    Recent work from several laboratories has demonstrated that proteolytic mechanisms significantly contribute to the molecular interplay between Streptococcus pyogenes, an important human pathogen, and its host. Here we describe the identification, purification and characterization of a novel extracellular cysteine proteinase produced by S.pyogenes. This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme of S.pyogenes, is distinct from the well-characterized streptococcal cysteine proteinase, SpeB, and cleaves human IgG in the hinge region with a high degree of specificity. Thus, other human proteins, including immunoglobulins M, A, D and E, are not degraded by IdeS. The enzyme efficiently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing of S.pyogenes by phagocytic cells. This and additional observations on the distribution and expression of the ideS gene indicate that IdeS represents a novel and significant bacterial virulence determinant, and a potential therapeutic target. PMID:11927545

  15. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G

    PubMed Central

    von Pawel-Rammingen, Ulrich; Johansson, Björn P.; Björck, Lars

    2002-01-01

    Recent work from several laboratories has demonstrated that proteolytic mechanisms significantly contribute to the molecular interplay between Streptococcus pyogenes, an important human pathogen, and its host. Here we describe the identification, purification and characterization of a novel extracellular cysteine proteinase produced by S.pyogenes. This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme of S.pyogenes, is distinct from the well-characterized streptococcal cysteine proteinase, SpeB, and cleaves human IgG in the hinge region with a high degree of specificity. Thus, other human proteins, including immunoglobulins M, A, D and E, are not degraded by IdeS. The enzyme efficiently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing of S.pyogenes by phagocytic cells. This and additional observations on the distribution and expression of the ideS gene indicate that IdeS represents a novel and significant bacterial virulence determinant, and a potential therapeutic target. PMID:11927545

  16. Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

    PubMed

    Müller, A; Benoist, P; Diem, H G; Schwencke, J

    1991-12-01

    To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest. PMID:15101385

  17. Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

    PubMed

    Pszenny, V; Angel, S O; Duschak, V G; Paulino, M; Ledesma, B; Yabo, M I; Guarnera, E; Ruiz, A M; Bontempi, E J

    2000-04-15

    A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host. PMID:10779600

  18. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    PubMed

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%. PMID:26304452

  19. A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.

    PubMed

    Moss, Marcia L; Koller, Garrit; Bartsch, Jörg W; Rakow, Sinja; Schlomann, Uwe; Rasmussen, Fred H

    2015-09-01

    We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity. PMID:26026386

  20. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

    PubMed Central

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-01-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  1. Electro-optic imaging Fourier transform spectrometer

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin (Inventor); Znod, Hanying (Inventor)

    2009-01-01

    An Electro-Optic Imaging Fourier Transform Spectrometer (EOIFTS) for Hyperspectral Imaging is described. The EOIFTS includes an input polarizer, an output polarizer, and a plurality of birefringent phase elements. The relative orientations of the polarizers and birefringent phase elements can be changed mechanically or via a controller, using ferroelectric liquid crystals, to substantially measure the spectral Fourier components of light propagating through the EIOFTS. When achromatic switches are used as an integral part of the birefringent phase elements, the EIOFTS becomes suitable for broadband applications, with over 1 micron infrared bandwidth.

  2. A Fourier approach to cloud motion estimation

    NASA Technical Reports Server (NTRS)

    Arking, A.; Lo, R. C.; Rosenfield, A.

    1977-01-01

    A Fourier technique is described for estimating cloud motion from pairs of pictures using the phase of the cross spectral density. The method allows motion estimates to be made for individual spatial frequencies, which are related to cloud pattern dimensions. Results obtained are presented and compared with the results of a Fourier domain cross correlation scheme. Using both artificial and real cloud data show that the technique is relatively sensitive to the presence of mixtures of motions, changes in cloud shape, and edge effects.

  3. Fast Fourier Transform algorithm design and tradeoffs

    NASA Technical Reports Server (NTRS)

    Kamin, Ray A., III; Adams, George B., III

    1988-01-01

    The Fast Fourier Transform (FFT) is a mainstay of certain numerical techniques for solving fluid dynamics problems. The Connection Machine CM-2 is the target for an investigation into the design of multidimensional Single Instruction Stream/Multiple Data (SIMD) parallel FFT algorithms for high performance. Critical algorithm design issues are discussed, necessary machine performance measurements are identified and made, and the performance of the developed FFT programs are measured. Fast Fourier Transform programs are compared to the currently best Cray-2 FFT program.

  4. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    PubMed Central

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  5. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    PubMed

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  6. Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase.

    PubMed

    Vacca, R A; Giannattasio, S; Graber, R; Sandmeier, E; Marra, E; Christen, P

    1997-08-29

    Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct. PMID:9268327

  7. Implementation of quantum and classical discrete fractional Fourier transforms

    PubMed Central

    Weimann, Steffen; Perez-Leija, Armando; Lebugle, Maxime; Keil, Robert; Tichy, Malte; Gräfe, Markus; Heilmann, René; Nolte, Stefan; Moya-Cessa, Hector; Weihs, Gregor; Christodoulides, Demetrios N.; Szameit, Alexander

    2016-01-01

    Fourier transforms, integer and fractional, are ubiquitous mathematical tools in basic and applied science. Certainly, since the ordinary Fourier transform is merely a particular case of a continuous set of fractional Fourier domains, every property and application of the ordinary Fourier transform becomes a special case of the fractional Fourier transform. Despite the great practical importance of the discrete Fourier transform, implementation of fractional orders of the corresponding discrete operation has been elusive. Here we report classical and quantum optical realizations of the discrete fractional Fourier transform. In the context of classical optics, we implement discrete fractional Fourier transforms of exemplary wave functions and experimentally demonstrate the shift theorem. Moreover, we apply this approach in the quantum realm to Fourier transform separable and path-entangled biphoton wave functions. The proposed approach is versatile and could find applications in various fields where Fourier transforms are essential tools. PMID:27006089

  8. Implementation of quantum and classical discrete fractional Fourier transforms

    NASA Astrophysics Data System (ADS)

    Weimann, Steffen; Perez-Leija, Armando; Lebugle, Maxime; Keil, Robert; Tichy, Malte; Gräfe, Markus; Heilmann, René; Nolte, Stefan; Moya-Cessa, Hector; Weihs, Gregor; Christodoulides, Demetrios N.; Szameit, Alexander

    2016-03-01

    Fourier transforms, integer and fractional, are ubiquitous mathematical tools in basic and applied science. Certainly, since the ordinary Fourier transform is merely a particular case of a continuous set of fractional Fourier domains, every property and application of the ordinary Fourier transform becomes a special case of the fractional Fourier transform. Despite the great practical importance of the discrete Fourier transform, implementation of fractional orders of the corresponding discrete operation has been elusive. Here we report classical and quantum optical realizations of the discrete fractional Fourier transform. In the context of classical optics, we implement discrete fractional Fourier transforms of exemplary wave functions and experimentally demonstrate the shift theorem. Moreover, we apply this approach in the quantum realm to Fourier transform separable and path-entangled biphoton wave functions. The proposed approach is versatile and could find applications in various fields where Fourier transforms are essential tools.

  9. Photonic quasi-crystals in Fourier and Fourier-Bessel space

    NASA Astrophysics Data System (ADS)

    Newman, S. R.; Gauthier, R. C.

    2013-02-01

    Photonic crystals that are aperiodic or quasi-crystalline in nature have been the focus of research due to their complex spatial distributions, resulting in high order rotational symmetries. Recently we proposed aperiodic patterns that were rotationally symmetric while being random in the radial direction. The structures are designed by segmenting the circular design space, randomly populating one segment, and repeating that segment about a center of rotation. Studying the symmetries and geometrical attributes of aperiodic structures is typically performed in reciprocal Fourier space by examining the distribution of the Fourier coefficients. This allows the translational symmetry to be directly extracted and the rotational nature to be interpreted. Instead we propose comparing the typical Fourier analysis with the use of a Fourier-Bessel space. The Fourier-Bessel approach expands the dielectric layout in cylindrical coordinates using exponential and Bessel functions as the angular and radial basis functions. The coefficients obtained in this fashion directly provide the rotational symmetries that are present. This work will examine both the Fourier and Fourier-Bessel distributions of the proposed structures as well as other quasi-crystals in order to explore the strengths and weaknesses of both techniques.

  10. Geometric Representations for Discrete Fourier Transforms

    NASA Technical Reports Server (NTRS)

    Cambell, C. W.

    1986-01-01

    Simple geometric representations show symmetry and periodicity of discrete Fourier transforms (DFT's). Help in visualizing requirements for storing and manipulating transform value in computations. Representations useful in any number of dimensions, but particularly in one-, two-, and three-dimensional cases often encountered in practice.

  11. Ultrafast Fourier-transform parallel processor

    SciTech Connect

    Greenberg, W.L.

    1980-04-01

    A new, flexible, parallel-processing architecture is developed for a high-speed, high-precision Fourier transform processor. The processor is intended for use in 2-D signal processing including spatial filtering, matched filtering and image reconstruction from projections.

  12. Fourier technique for studying ammonoid sutures

    SciTech Connect

    Gildner, R.F.; Ackerly, S.C.

    1985-01-01

    Suture patterns have long been recognized as being of primary importance in the study of ammonoids. The authors have developed a technique to use Fourier analysis to study these structures by using a simple transformation: x-y data of a digitized suture are transformed to angle of slope versus position along the suture's length. A Fast Fourier Transform applied to the data produces a power spectrum (amplitude versus wave number) providing a precise and accurate measure of suture shape. The authors have applied this technique to the analysis of ontogenetic change in suture morphology. In goniatitic, ceratitic and preadult ammonitic patterns most of the change is exhibited in the amplitudes of the lowest ten wave numbers. Their Fourier coefficients clearly show trends not readily apparent by visual inspection. The more complex ammonitic patterns are reflected in increased amplitudes of higher wave numbers (a broader peak of the power spectrum) and their analysis is necessarily more complex. The Fourier approach presents the opportunity to quantitatively measure and describe the tempo and mode of evolution in the Ammonoidea. Potential applications of the new technique, as well as limitations, are discussed with special attention to investigations of ammonoid ontogeny and phylogeny.

  13. Clifford Fourier transform on vector fields.

    PubMed

    Ebling, Julia; Scheuermann, Gerik

    2005-01-01

    Image processing and computer vision have robust methods for feature extraction and the computation of derivatives of scalar fields. Furthermore, interpolation and the effects of applying a filter can be analyzed in detail and can be advantages when applying these methods to vector fields to obtain a solid theoretical basis for feature extraction. We recently introduced the Clifford convolution, which is an extension of the classical convolution on scalar fields and provides a unified notation for the convolution of scalar and vector fields. It has attractive geometric properties that allow pattern matching on vector fields. In image processing, the convolution and the Fourier transform operators are closely related by the convolution theorem and, in this paper, we extend the Fourier transform to include general elements of Clifford Algebra, called multivectors, including scalars and vectors. The resulting convolution and derivative theorems are extensions of those for convolution and the Fourier transform on scalar fields. The Clifford Fourier transform allows a frequency analysis of vector fields and the behavior of vector-valued filters. In frequency space, vectors are transformed into general multivectors of the Clifford Algebra. Many basic vector-valued patterns, such as source, sink, saddle points, and potential vortices, can be described by a few multivectors in frequency space. PMID:16138556

  14. A Primer of Fourier Transform NMR.

    ERIC Educational Resources Information Center

    Macomber, Roger S.

    1985-01-01

    Fourier transform nuclear magnetic resonance (NMR) is a new spectroscopic technique that is often omitted from undergraduate curricula because of lack of instructional materials. Therefore, information is provided to introduce students to the technique of data collection and transformation into the frequency domain. (JN)

  15. REMOTE FOURIER TRANSFORM INFRARED AIR POLLUTION STUDIES

    EPA Science Inventory

    A commercial Fourier transform infrared interferometer system has been installed in a van and used to make longpath absorption and single-ended emission measurements of gaseous pollutant concentrations at a variety of pollutant sources. The interferometer system is described and ...

  16. Destriping AIS data using Fourier filtering techniques

    NASA Technical Reports Server (NTRS)

    Hlavka, C.

    1986-01-01

    Airborne Imaging Spectrometers (AIS) data collected in 1984 and 1985 showed pronounced striping in the vertical and horizontal directions. This striping reduced the signal to noise ratio so that features of the spectra of forest canopies were obscured or altered by noise. This noise was removed by application of a notch filter to the Fourier transform of the imagery in each waveband.

  17. Livermore Imaging Fourier Transform Infrared Spectrometer (LIFTIRS)

    SciTech Connect

    Carter, M.R.; Bennett, C.L.; Fields, D.J.; Lee, F.D.

    1995-05-10

    Lawrence Livermore National Laboratory is currently operating a hyperspectral imager, the Livermore Imaging Fourier Transform Infrared Spectrometer (LIFTIRS). This instrument is capable of operating throughout the infrared spectrum from 3 to 12.5 {mu}m with controllable spectral resolution. In this presentation we report on it`s operating characteristics, current capabilities, data throughput and calibration issues.

  18. Fourier theory and C∗-algebras

    NASA Astrophysics Data System (ADS)

    Bédos, Erik; Conti, Roberto

    2016-07-01

    We discuss a number of results concerning the Fourier series of elements in reduced twisted group C∗-algebras of discrete groups, and, more generally, in reduced crossed products associated to twisted actions of discrete groups on unital C∗-algebras. A major part of the article gives a review of our previous work on this topic, but some new results are also included.

  19. Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis.

    PubMed

    Yang, Qingzhu; Yu, Kun; Yan, Liming; Li, Yuanyuan; Chen, Cheng; Li, Xuemei

    2011-09-01

    The aspartate kinase (AK) from Mycobacterium tuberculosis (Mtb) catalyzes the biosynthesis of aspartate family amino acids, including lysine, threonine, isoleucine and methionine. We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone (referred to as MtbAKβ) and in complex with threonine (referred to as MtbAKβ-Thr) at resolutions of 2.6 Å and 2.0 Å, respectively. MtbAKβ is composed of two perpendicular non-equivalent ACT domains [aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase)] per monomer. Each ACT domain contains two α helices and four antiparallel β strands. The structure of MtbAKβ shares high similarity with the regulatory subunit of the aspartate kinase from Corynebacterium glutamicum (referred to as CgAKβ), suggesting similar regulatory mechanisms. Biochemical assays in our study showed that MtbAK is inhibited by threonine. Based on crystal structure analysis, we discuss the regulatory mechanism of MtbAK. PMID:21976064

  20. Interaction between L-aspartate and the brucite [Mg(OH)2]-water interface

    NASA Astrophysics Data System (ADS)

    Estrada, Charlene F.; Sverjensky, Dimitri A.; Pelletier, Manuel; Razafitianamaharavo, Angélina; Hazen, Robert M.

    2015-04-01

    The interaction of biomolecules at the mineral-water interface could have played a prominent role in the emergence of more complex organic species in life's origins. Serpentinite-hosted hydrothermal vents may have acted as a suitable environment for this process to occur, although little is known about biomolecule-mineral interactions in this system. We used batch adsorption experiments and surface complexation modeling to study the interaction of L-aspartate onto a thermodynamically stable product of serpentinization, brucite [Mg(OH)2], over a wide range of initial aspartate concentrations at four ionic strengths governed by [Mg2+] and [Ca2+]. We observed that up to 1.0 μmol of aspartate adsorbed per m2 of brucite at pH ∼ 10.2 and low Mg2+ concentrations (0.7 × 10-3 M), but surface adsorption decreased at high Mg2+ concentrations (5.8 × 10-3 M). At high Ca2+ concentrations (4.0 × 10-3 M), aspartate surface adsorption doubled (to 2.0 μmol m-2), with Ca2+ adsorption at 29.6 μmol m-2. We used the extended triple-layer model (ETLM) to construct a quantitative thermodynamic model of the adsorption data. We proposed three surface reactions involving the adsorption of aspartate (HAsp-) and/or Ca2+ onto brucite:

  1. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds. PMID:26254042

  2. Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

    PubMed Central

    Marra, E; Passarella, S; Casamassima, E; Perlino, E; Doonan, S; Quagliariello, E

    1985-01-01

    Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system. PMID:4015628

  3. Uptake and metabolism of (14C)-aspartate by developing kernels of maize (Zea mays L. )

    SciTech Connect

    Muhitch, M.J. )

    1990-05-01

    Pulse-chase experiments were performed to determine the metabolic fate of (14C)-aspartate in the pedicel region and subsequent uptake into the endosperm. Kernels were removed from the cob, leaving the pedicel attached but removing glumes, palea, and lemma. The basal tips were incubated in (14C)-aspartate for 0.5 h, followed by a 2 h chase period with unlabeled aspartate. In contrast to a previous study in which 70% of the 14C from aspartate was recovered in the organic acid fraction (Lyznik, et al., Phytochemistry 24: 425, 1985), only 20 to 25% of the radioactivity found in the 2 h chase period. While a small amount of the 14C transiently appeared in alanine at the beginning of the chase period, the most heavily labeled non-fed amino acid was glutamine, which accounted for 21% of the radioactivity within the pedicel amino acid fraction by 0.5 h into the chase period. There was no evidence for asparagine synthesis within the pedicel region of the kernel. 14C recovered from the endosperm in the form of amino acids were aspartate (60%), glutamine (20%), glutamate (15%), and alanine (5%). These results suggest that some of the maternally supplied amino acids undergo metabolic conversion to other amino acids before being taken up by the endosperm.

  4. Modification of the proteinase/anti-proteinase balance in the respiratory tract of Sprague-Dawley rats after single intratracheal instillation of benzo[A]pyrene-coated onto Fe(2)O(3) particles.

    PubMed

    Garçon, G; Campion, J; Hannothiaux, M H; Boutin, A C; Venembre, P; Balduyck, M; Haguenoer, J M; Shirali, P

    2000-01-01

    Available data suggest that repeated concurrent exposure to haematite (Fe(2)O(3)) and benzo[A]pyrene (B[A]P) results in a decreased latency and an increased incidence of lung tumours in rodents compared to exposure to B[A]P alone. Moreover, the reactive oxygen species (ROS) formed by the lung cells themselves and/or by activated inflammatory cells may possibly contribute to the development of pulmonary disorders such as cancer formation. In order to investigate the precise role of iron in the injury induced by B[A]P-coated onto Fe(2)O(3) particles, we tend to address the hypothesis that Fe(2)O(3) and B[A]P, alone or in association, can induce oxidative stress conditions (malondialdehyde) and/or inflammatory reactions (interleukin-6) and thereby disrupt the proteinase/anti-proteinase balance (cathepsins B and L, polynuclear neutrophil (PNN) elastase, alpha-1 proteinase inhibitor (alpha(1)PI) and its inhibitory capacity) in the rat respiratory tract. Thus, Fe(2)O(3) or B[A]P-coated onto Fe(2)O(3) particles produce oxidative stress conditions through not only iron-catalysed oxidative reactions but also inflammatory processes. However, B[A]P initiates only inflammatory responses. These pollutants generate increased levels of proteases and decrease the concentrations of free alpha(1)PI. There is also a clear relationship between the partial inactivation of alpha(1)PI and the occurrence of ROS after exposure to Fe(2)O(3), alone or as a carrier of B[A]P. Hence, the proteinase/anti-proteinase balance might be more disrupted by Fe(2)O(3) or B[A]P-coated onto Fe(2)O(3) particles than by B[A]P alone. These results suggest a mechanism that can explain why B[A]P-coated onto Fe(2)O(3) particles are more injurious than B[A]P alone. PMID:10942902

  5. Nonlinear Fourier analysis with cnoidal waves

    SciTech Connect

    Osborne, A.R.

    1996-12-31

    Fourier analysis is one of the most useful tools to the ocean engineer. The approach allows one to analyze wave data and thereby to describe a dynamical motion in terms of a linear superposition of ordinary sine waves. Furthermore, the Fourier technique allows one to compute the response function of a fixed or floating structure: each sine wave in the wave or force spectrum yields a sine wave in the response spectrum. The counting of fatigue cycles is another area where the predictable oscillations of sine waves yield procedures for the estimation of the fatigue life of structures. The ocean environment, however, is a source of a number of nonlinear effects which must also be included in structure design. Nonlinearities in ocean waves deform the sinusoidal shapes into other kinds of waves such as the Stokes wave, cnoidal wave or solitary wave. A key question is: Does there exist a generalization of linear Fourier analysis which uses nonlinear basis functions rather than the familiar sine waves? Herein addresses the dynamics of nonlinear wave motion in shallow water where the basis functions are cnoidal waves and discuss nonlinear Fourier analysis in terms of a linear superposition of cnoidal waves plus their mutual nonlinear interactions. He gives a number of simple examples of nonlinear Fourier wave motion and then analyzes an actual surface-wave time series obtained on an offshore platform in the Adriatic Sea. Finally, he briefly discusses application of the cnoidal wave spectral approach to the computation of the frequency response function of a floating vessel. The results given herein will prove useful in future engineering studies for the design of fixed, floating and complaint offshore structures.

  6. Effects of cysteine proteinase inhibitors scN and E-64 on southern corn rootworm larval development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The southern corn rootworm (SCRW) can be a serious pest of peanut pods. A laboratory bioassay was developed to test feeding cysteine proteinase inhibitors soyacystatin N (scN) and E-64 against southern corn rootworm reared on artificial diet to determine the effects on larvae development and mortal...

  7. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  8. Antisense Inhibition of Expression of Cysteine Proteinases Affects Entamoeba histolytica-Induced Formation of Liver Abscess in Hamsters

    PubMed Central

    Ankri, Serge; Stolarsky, Tamara; Bracha, Rivka; Padilla-Vaca, Felipe; Mirelman, David

    1999-01-01

    Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters. PMID:9864246

  9. Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids.

    PubMed

    Bachovchin, W W; Plaut, A G; Flentke, G R; Lynch, M; Kettner, C A

    1990-03-01

    The alpha-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes. PMID:2105953

  10. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    PubMed

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. PMID:25043896

  11. Mapping of the Proteinase B Structural Gene PRB1, in SACCHAROMYCES CEREVISIAE and Identification of Nonsense Alleles within the Locus

    PubMed Central

    Zubenko, George S.; Mitchell, Aaron P.; Jones, Elizabeth W.

    1980-01-01

    We report the mapping of the structural gene for proteinase B, PRB1. It is located 1.1 cM proximal to CAN1 on the left arm of chromosome V of Saccharomyces cerevisiae. We have identified 34 amber and 12 ochre mutations among the 126 prb1 mutations in our collection. PMID:7009321

  12. MANGANESE UPREGULATES CELLULAR PRION PROTEINS AND INHIBITS THE RATE OF PROTEINASE-K DEPENDENT LIMITED PROTEOLYSIS IN NEURONAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The key event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent cations such as copper to th...

  13. 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

  14. A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase.

    PubMed

    Fook, J M S L L; Macedo, L L P; Moura, G E D D; Teixeira, F M; Oliveira, A S; Queiroz, A F S; Sales, M P

    2005-05-01

    Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors. PMID:15820500

  15. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K

    PubMed Central

    Ali Mohammed, Marwan Mansoor; Nerland, Audun H.; Al-Haroni, Mohammed; Bakken, Vidar

    2013-01-01

    Background Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. Methods F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Results Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. Conclusion DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions. PMID:23372876

  16. Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants1

    PubMed Central

    De Leo, Francesca; Bonadé-Bottino, Michel A.; Ceci, Luigi R.; Gallerani, Raffaele; Jouanin, Lise

    1998-01-01

    This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI. PMID:9808744

  17. 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

  18. Serum Insulin Aspart Concentrations Following High-Dose Insulin Aspart Administered Directly into the Duodenum of Healthy Subjects: An Open-Labeled, Single-Blinded, and Uncontrolled Exploratory Trial

    PubMed Central

    Ihlo, Charlotte A.; Aksglæde, Karin Bak; Laursen, Torben; Lauritzen, Torsten; Christiansen, Jens Sandahl

    2009-01-01

    Objective The goal of this study was to determine the bioavailability of high-dose insulin aspart administered directly into the duodenum of healthy subjects. Methods In a pilot study, four subjects each received four escalating doses of a 1-ml solution of insulin aspart (100, 300, 600, and 1000 IU, respectively) directly into the duodenum. In the following main study, eight subjects each received two identical doses of insulin aspart of 1000 IU, in 4- and 8-ml solutions, respectively, directly into the duodenum. Subjects in the main study also received an intravenous and a subcutaneous injection of 4 to 6 IU of insulin aspart. Results A considerable number of samples and, in some cases, consecutive samples revealed significantly increased concentrations of serum insulin aspart. Despite the significant serum insulin aspart concentrations, no significant changes of plasma glucose were measured. Moreover, no significant suppression of endogenous insulin secretion was detected, as assessed by the levels of serum human insulin. Conclusions Administration of high-dose insulin aspart directly into the duodenum of healthy subjects resulted in significantly increased serum insulin aspart concentrations in a high number of consecutive samples using a specific enzyme-linked immunosorbent assay. However, no significant changes in the levels of plasma glucose or serum human insulin were observed. Thus, the study did not provide any evidence of biological activity of the original insulin aspart molecule after high-dose administration directly into the duodenum. PMID:20144435

  19. Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59.

    PubMed Central

    Lu, Y; Lu, X; Denison, M R

    1995-01-01

    Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, and E. V. Koonin, FEBS Lett. 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res. 17:4847-4861, 1989). We have cloned into a T7 transcription vector a cDNA fragment containing the putative 3C-like proteinase domain of MHV-A59, along with portions of the flanking hydrophobic domains. The construct was used to express a polypeptide in a combined in vitro transcription-translation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecular masses of 32 and 27 kDa were predominant after 30 to 45 min and appeared to be products of specific proteolysis of larger precursors. Mutations at the putative catalytic histidine and cysteine residues abolished the processing of the 27-kDa protein. Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine mutants. The amino-terminal cleavage of the 27-kDa protein occurred at a glutamine-serine dipeptide as previously predicted. This study provides experimental confirmation that the coronaviruses express an active proteinase within the 3C-like proteinase domain of gene 1 ORF 1a and that this proteinase utilizes at least one canonical QS dipeptide as a cleavage site in vitro. PMID:7745703

  20. The drug 5-aminosalicylic acid rescues alpha 1-proteinase inhibitor from the neutrophil oxidative inactivation. A possible contribution to its therapeutic action in ulcerative colitis.

    PubMed

    Ottonello, L; Dapino, P; Pastorino, G; Vitale, E; Dallegri, F

    1992-01-01

    The glycoprotein alpha 1-proteinase inhibitor is the specific inhibitor of neutrophil elastase, a major tissue-damaging protease. When incubated with activated neutrophils, alpha 1-proteinase inhibitor lost its pancreatic porcine elastase inhibitory capacity and became incapable of forming a sodium dodecyl sulphate-stable complex with pancreatic porcine elastase. Inhibitors and scavengers of neutrophil-derived reactive oxygen species outlined the crucial role of hypochlorous acid in the alpha 1-proteinase inhibitor inactivation. Moreover, the drug 5-aminosalicylic acid prevented the inactivation of alpha 1-proteinase inhibitor by neutrophils in a dose-dependent manner. Finally, when the capacity of 5-aminosalicylic acid to rescue alpha 1-proteinase inhibitor from the neutrophil-derived attack was plotted as a function of the 5-aminosalicylic acid ability to scavenge neutrophil-derived hypochlorous acid, a positive linear relationship was found. Thus, our results provide a direct evidence that 5-aminosalicylic acid is able to prevent the oxidative inactivation of alpha 1-proteinase inhibitor by neutrophils. Therefore, we suggest that the drug has the potential to limit the elastase-mediated damage of colonic connective tissue by creating a microenvironment of active alpha 1-proteinase inhibitor around the neutrophils. PMID:1521714

  1. A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

    PubMed

    Koiwa, H; Shade, R E; Zhu-Salzman, K; D'Urzo, M P; Murdock, L L; Bressan, R A; Hasegawa, P M

    2000-04-01

    Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN. PMID:10760514

  2. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed Central

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-01-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  3. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-11-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  4. Surface Zn-proteinase as a molecule for defense of Leishmania mexicana amazonensis promastigotes against cytolysis inside macrophage phagolysosomes.

    PubMed Central

    Seay, M B; Heard, P L; Chaudhuri, G

    1996-01-01

    The role of the surface membrane Zn-proteinase in protecting the cellular integrity of the macrophage parasite Leishmania mexicana amazonensis from intraphagolysosomal cytolysis was studied. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth medium. The virulent and attenuated variants of the parasite cells were cloned. Failure of these attenuated parasite cells to survive inside macrophage phagolysosomes is associated with 20- to 50-fold reduction in the expression of surface gp63 protein. In situ inhibition of gp63 proteinase activity inside Leishmania-infected macrophage phagolysosomes with targeted delivery of an inhibitor of gp63 proteinase activity, 1,10-phenanthroline, selectively eliminated intracellular Leishmania amastigotes, further suggesting the importance of this proteinase in phagolysosomal survival of the parasite. An upstream sequence (US) of the gp63 gene was cloned in front of the bacterial chloramphenicol acetyltransferase (CAT) gene in plasmid pCATbasic. Transfection of L. mexicana amazonensis cells with this recombinant plasmid showed that expression of the CAT gene from this US is 15- to 20-fold higher in virulent clones than in avirulent clones of the parasite. Band shift analysis with the cloned US also showed that binding of protein(s) was 15- to 20-fold higher in virulent cell extract than in avirulent cell extract. Coating of attenuated cells or liposomes with proteolytically active gp63 protects them from degradation inside macrophage phagolysosomes. These results suggest a novel mechanism of survival of this phagolysosomal parasite with the help of its surface Zn-proteinase. PMID:8945556

  5. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut

    PubMed Central

    Miller, Hugh R P; Pemberton, Alan D

    2002-01-01

    Serine proteinases with trypsin-like (tryptase) and chymotrypsin-like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa-specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa-specific chymase, mouse mast cell proteinase-1 (mMCP-1), is regulated by transforming growth factor-β1 (TGF-β1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase-activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin-I to angiotensin-II. Mucosa-specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in

  6. Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

    PubMed Central

    Azanza, Jean-Louis; Raymond, Jacques; Robin, Jean-Michel; Cottin, Patrick; Ducastaing, André

    1979-01-01

    Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase. ImagesFig. 1.Fig. 2.Fig. 3. PMID:534501

  7. N-phosphonacetyl-L-isoasparagine a Potent and Specific Inhibitor of E. coli Aspartate Transcarbamoylase†

    PubMed Central

    Eldo, Joby; Cardia, James P.; O’Day, Elizabeth M.; Xia, Jiarong; Tsurata, Hiro; Kantrowitz, Evan R.

    2008-01-01

    The synthesis of a new inhibitor, N-phosphonacetyl-L-isoasparagine (PALI), of Escherichia coli aspartate transcarbamoylase (ATCase) is reported, as well as structural studies of the enzyme·PALI complex. PALI was synthesized in 7 steps from β-benzyl L-aspartate. The KD of PALI was 2 μM. Kinetics and small-angle X-ray scattering experiments showed that PALI can induce the cooperative transition of ATCase from the T to the R state. The X-ray structure of the enzyme·PALI complex showed 22 hydrogen bonding interactions between the enzyme and PALI. The kinetic characterization and crystal structure of the ATCase·PALI complex also provides detailed information regarding the importance of the α-carboxylate for the binding of the substrate aspartate. PMID:17004708

  8. Action of aspartate on the /sup 32/Pi incorporation into phospholipids of cerebral cortex

    SciTech Connect

    de Scarnati, O.C.; Sato, M.; De Robertis, E.

    1982-02-01

    The effect of L-aspartate on the /sup 32/Pi incorporation of phospholipids, was studied on slices of rat cerebral cortex. This amino acid produced an inhibitory effect in concentrations 0.01-10 mM, which was more evident at 120 min. This effect was not stereospecific and did not imply a change in Pi uptake and in nucleotides approximating P precursors. The inhibition was present in PS, PC, PE and to a lesser extent in Pi. On liver slices 1 mM L-aspartate had the opposite effect, stimulating the incorporation of /sup 32/Pi into total phospholipids. Our results suggest that the effect of L-aspartate is by a non-specific mechanism, probably not mediated by a receptor.

  9. Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

    PubMed Central

    Marra, E; Doonan, S; Saccone, C; Quagliariello, E

    1977-01-01

    1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo. PMID:883959

  10. Adsorption of L-aspartate to rutile (α-TiO 2): Experimental and theoretical surface complexation studies

    NASA Astrophysics Data System (ADS)

    Jonsson, Caroline M.; Jonsson, Christopher L.; Estrada, Charlene; Sverjensky, Dimitri A.; Cleaves, H. James, II; Hazen, Robert M.

    2010-04-01

    Interactions between aqueous amino acids and mineral surfaces influence many geochemical processes from biomineralization to the origin of life. However, the specific reactions involved and the attachment mechanisms are mostly unknown. We have studied the adsorption of L-aspartate on the surface of rutile (α-TiO 2, pH PPZC = 5.4) in NaCl(aq) over a wide range of pH, ligand-to-solid ratio and ionic strength, using potentiometric titrations and batch adsorption experiments. The adsorption is favored below pH 6 with a maximum of 1.2 μmol of adsorbed aspartate per m 2 of rutile at pH 4 in our experiments. The adsorption decreases at higher pH because the negatively charged aspartate molecule is repelled by the negatively charged rutile surface above pH PPZC. At pH values of 3-5, aspartate adsorption increases with decreasing ionic strength. The adsorption of aspartate on rutile is very similar to that previously published for glutamate ( Jonsson et al., 2009). An extended triple-layer model was used to provide a quantitative thermodynamic characterization of the aspartate adsorption data. Two reaction stoichiometries identical in reaction stoichiometry to those for glutamate were needed. At low surface coverages, aspartate, like glutamate, may form a bridging-bidentate surface species binding through both carboxyl groups, i.e. "lying down" on the rutile surface. At high surface coverages, the reaction stoichiometry for aspartate was interpreted differently compared to glutamate: it likely involves an outer-sphere or hydrogen bonded aspartate surface species, as opposed to a partly inner-sphere complex for glutamate. Both the proposed aspartate species are qualitatively consistent with previously published ATR-FTIR spectroscopic results for aspartate on amorphous titanium dioxide. The surface complexation model for aspartate was tested against experimental data for the potentiometric titration of aspartate in the presence of rutile. In addition, the model correctly

  11. Herz spaces and restricted summability of Fourier transforms and Fourier series

    NASA Astrophysics Data System (ADS)

    Weisz, Ferenc

    2008-08-01

    A general summability method, the so-called [theta]-summability is considered for multi-dimensional Fourier transforms and Fourier series. A new inequality for the Hardy-Littlewood maximal function is verified. It is proved that if the Fourier transform of [theta] is in a Herz space, then the restricted maximal operator of the [theta]-means of a distribution is of weak type (1,1), provided that the supremum in the maximal operator is taken over a cone-like set. From this it follows that over a cone-like set a.e. for all . Moreover, converges to f(x) over a cone-like set at each Lebesgue point of if and only if the Fourier transform of [theta] is in a suitable Herz space. These theorems are extended to Wiener amalgam spaces as well. The Riesz and Weierstrass summations are investigated as special cases of the [theta]-summation.

  12. Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases.

    PubMed

    Wagstaff, Simon C; Harrison, Robert A

    2006-08-01

    Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A(2), C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved alpha(9)beta(1) integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases. PMID:16713134

  13. Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.

    PubMed Central

    Topham, C M; Salih, E; Frazao, C; Kowlessur, D; Overington, J P; Thomas, M; Brocklehurst, S M; Patel, M; Thomas, E W; Brocklehurst, K

    1991-01-01

    1. A model of the three-dimensional structure of papaya proteinase omega, the most basic cysteine proteinase component of the latex of papaya (Carica papaya), was built from its amino acid sequence and the two currently known high-resolution crystal structures of the homologous enzymes papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). The method used a knowledge-based approach incorporated in the COMPOSER suite of programs and refinement by using the interactive graphics program FRODO on an Evans and Sutherland PS 390 and by energy minimization using the GROMOS program library. 2. Functional similarities and differences between the three cysteine proteinases revealed by analysis of pH-dependent kinetics of the acylation process of the catalytic act and of the reactions of the enzyme catalytic sites with substrate-derived 2-pyridyl disulphides as two-hydronic-state reactivity probes are reported and discussed in terms of the knowledge-based model. 3. To facilitate analysis of complex pH-dependent kinetic data, a multitasking application program (SKETCHER) for parameter estimation by interactive manipulation of calculated curves and a simple method of writing down pH-dependent kinetic equations for reactions involving any number of reactive hydronic states by using information matrices were developed. 4. Papaya proteinase omega differs from the other two enzymes in the ionization characteristics of the common (Cys)-SH/(His)-Im+H catalytic-site system and of the other acid/base groups that modulate thiol reactivity towards substrate-derived inhibitors and the acylation process of the catalytic act. The most marked difference in the Cys/His system is that the pKa for the loss of the ion-pair state to form -S-/-Im is 8.1-8.3 for papaya proteinase omega, whereas it is 9.5 for both actinidin and papain. Papaya proteinase omega is similar to actinidin in that it lacks the second catalytically influential group with pKa approx. 4 present in papain and possesses a

  14. Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.

    PubMed

    Topham, C M; Salih, E; Frazao, C; Kowlessur, D; Overington, J P; Thomas, M; Brocklehurst, S M; Patel, M; Thomas, E W; Brocklehurst, K

    1991-11-15

    1. A model of the three-dimensional structure of papaya proteinase omega, the most basic cysteine proteinase component of the latex of papaya (Carica papaya), was built from its amino acid sequence and the two currently known high-resolution crystal structures of the homologous enzymes papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). The method used a knowledge-based approach incorporated in the COMPOSER suite of programs and refinement by using the interactive graphics program FRODO on an Evans and Sutherland PS 390 and by energy minimization using the GROMOS program library. 2. Functional similarities and differences between the three cysteine proteinases revealed by analysis of pH-dependent kinetics of the acylation process of the catalytic act and of the reactions of the enzyme catalytic sites with substrate-derived 2-pyridyl disulphides as two-hydronic-state reactivity probes are reported and discussed in terms of the knowledge-based model. 3. To facilitate analysis of complex pH-dependent kinetic data, a multitasking application program (SKETCHER) for parameter estimation by interactive manipulation of calculated curves and a simple method of writing down pH-dependent kinetic equations for reactions involving any number of reactive hydronic states by using information matrices were developed. 4. Papaya proteinase omega differs from the other two enzymes in the ionization characteristics of the common (Cys)-SH/(His)-Im+H catalytic-site system and of the other acid/base groups that modulate thiol reactivity towards substrate-derived inhibitors and the acylation process of the catalytic act. The most marked difference in the Cys/His system is that the pKa for the loss of the ion-pair state to form -S-/-Im is 8.1-8.3 for papaya proteinase omega, whereas it is 9.5 for both actinidin and papain. Papaya proteinase omega is similar to actinidin in that it lacks the second catalytically influential group with pKa approx. 4 present in papain and possesses a

  15. Aspartate Embedding Depth Affects pHLIP’s Insertion pKa

    PubMed Central

    Fendos, Justin; Barrera, Francisco N.; Engelman, Donald M.

    2014-01-01

    We have used the pHLIP® (pH Low Insertion Peptide) peptide family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To further examine this protonation mechanism, we created pHLIP sequence variants in which the position of the two spanning aspartates (D14, D25) was moved up or down in the sequence. We hypothesized that aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to see if arginine snorkeling modulates the insertion pKa by affecting aspartate depth. Our results indicate both types of mutations change the insertion pKa, supporting the idea that aspartate depth is a participating parameter in determining pH dependence. We also show that pHLIP’s resistance to aggregation can be altered with our mutations, identifying a new criterion for improving pHLIP performance in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  16. Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate

    SciTech Connect

    Mise, Takeshi; Matsunami, Hideyuki; Samatey, Fadel A.; Maruyama, Ichiro N.

    2014-08-27

    The periplasmic domain of the E. coli aspartate receptor Tar was cloned, expressed, purified and crystallized with and without bound ligand. The crystals obtained diffracted to resolutions of 1.58 and 1.95 Å, respectively. The cell-surface receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni{sup 2+}. To understand the molecular mechanisms underlying the induction of Tar activity by its ligands, the Escherichia coli Tar periplasmic domain with and without bound aspartate (Asp-Tar and apo-Tar, respectively) were each crystallized in two different forms. Using ammonium sulfate as a precipitant, crystals of apo-Tar1 and Asp-Tar1 were grown and diffracted to resolutions of 2.10 and 2.40 Å, respectively. Alternatively, using sodium chloride as a precipitant, crystals of apo-Tar2 and Asp-Tar2 were grown and diffracted to resolutions of 1.95 and 1.58 Å, respectively. Crystals of apo-Tar1 and Asp-Tar1 adopted space group P4{sub 1}2{sub 1}2, while those of apo-Tar2 and Asp-Tar2 adopted space groups P2{sub 1}2{sub 1}2{sub 1} and C2, respectively.

  17. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Qatar cohort of the A1chieve study

    PubMed Central

    Daghash, Mohamed Hasan; Raja, Jabbar Mubarak; Milad, Mohamed

    2013-01-01

    Background: The A1chieve, a multicentric (28 countries), 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726) in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Qatar. Results: A total of 91 patients were enrolled in the study. Two insulin analogue regimens were used in the study. Study patients had started on or were switched to biphasic insulin aspart (n = 88), insulin detemir (n = 2), and other insulin combinations (n = 1). At baseline glycaemic control was poor for both insulin naïve (mean HbA1c: 10.9%) and insulin users (mean HbA1c: 9.1%) groups. After 24 weeks of treatment, all the study groups showed improvement in HbA1c (insulin naïve: −1.8%, insulin users: −1.3%). Major hypoglycaemia did not occur in the study patients. SADRs were reported in 1.4% of insulin users. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. PMID:24404484

  18. Biospecific haemosorbents based on proteinase inhibitor. II. Efficiency of biospecific antiproteinase haemosorbent 'Ovosorb' in complex treatment of experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs.

    PubMed

    Platé, N A; Kirkovsky, V V; Antiperovich, O F; Nicolaichik, V V; Valueva, T A; Sinilo, S B; Moin, V M; Lobacheva, G A

    1994-03-01

    The biospecific antiproteinase haemosorbent (BAH) 'Ovosorb' containing, in the bulk of polyacryamide gel, the ovomucoid from whites of duck eggs, was used for a complex treatment of the experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs. The efficiency of BAH was manifested in the significant reduction of lethality of the experimental animals, a more rapid liquidation of proteinasaemia, normalization in plasma of alpha 1-proteinase inhibitor and protein metabolism. Thus, by eliminating proteinases from circulation, Ovosorb contributes to the cessation of imbalance in the proteinase-inhibitor system and is efficient in the therapy of pathological states related to this imbalance. PMID:8031989

  19. Purification and characterization of elastase-specific inhibitor. Sequence homology with mucus proteinase inhibitor.

    PubMed

    Sallenave, J M; Ryle, A P

    1991-01-01

    Elastase-specific inhibitor (ESI) was purified from sputum of patients with chronic bronchitis and compared with mucus proteinase inhibitor (MPI, BrI) isolated, without the use of affinity chromatography on an enzyme, from non-purulent sputum of a patient with bronchial carcinoma. The N-terminal sequence of 27 residues of the latter was determined and showed serine as the only N-terminus. The partial N-terminal amino-acid sequence of ESI shows some homology with MPI, especially around the reactive site of MPI for human neutrophil elastase. This region could therefore be the reactive site of ESI. The thermodynamic and kinetic constants of the reactions of ESI with human neutrophil elastase and with porcine pancreatic elastase show that ESI is a fast-acting inhibitor. PMID:2039600

  20. The evolution of a genetic locus encoding small serine proteinase inhibitors ⋆

    PubMed Central

    Clauss, Adam; Lilja, Hans; Lundwall, Åke

    2007-01-01

    We previously identified a locus on human chromosome 20 that encompasses 14 genes of postulated WFDC-type proteinase inhibitors with a potential role in innate immunity. In an extended study, homologous loci are here described on mouse chromosome 2, rat chromosome 3, and dog chromosome 24. As in humans, the murine and canine loci are divided into two sub-loci separated by 0.2 Mb. The majority of genes are conserved in all species, but there are also species-specific gains and losses of genes, e.g., several duplications have yielded four SLPI genes in the rat and, most surprisingly, there is no murine elafin gene. Two human pseudogenes were identified due to the discovery of functional rodent genes. The conservation of different WFDC domains varies considerably, and it is hypothesized that this reflects a dual role of WFDC inhibitors in natural immunity, which is directed both against microbes and proinflammatory cells. PMID:15950183

  1. Alpha 1-proteinase inhibitor is more sensitive to inactivation by cigarette smoke than is leukocyte elastase

    SciTech Connect

    Janoff, A.; Dearing, R.

    1982-10-01

    Aqueous solutions of gas phase cigarette smoke were incubated with pure human leukocyte elastase or with crude human leukocyte granule extract, and the effects on enzyme activity were determined using a synthetic amide substrate. Simultaneously, the same smoke solutions were incubated with 10% human serum under identical conditions, and the effects on serum inhibition of purified or crude leukocyte elastase were similarly measured. In addition, aqueous solutions of unfractionated cigarette smoke were incubated with leukocyte elastase or serum, and the abilities of the smoke-treated enzyme to digest elastin and of the smoke-treated serum to inhibit elastin digestion were determined. Both experimental protocols showed that serum elastase-inhibiting capacity (primarily caused by alpha 1-proteinase inhibitor) is more susceptible to inactivation by aqueous solutions of cigarette smoke than is leukocyte elastase, suggesting that elastase inhibition (rather than elastase activity) may be predominantly suppressed by cigarette smoke inhalation in vivo.

  2. Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

    PubMed

    Di Pierro, Giovanna; O'Keeffe, Martina B; Poyarkov, Alexey; Lomolino, Giovanna; FitzGerald, Richard J

    2014-08-01

    A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and β-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19μmol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the β-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that β-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h β-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties. PMID:24629973

  3. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-01

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections. PMID:25881537

  4. Specificity of the collagenolytic serine proteinase from the pancreas of the catfish (Parasilurus asotus).

    PubMed

    Yoshinaka, R; Sato, M; Yamashita, M; Itoko, M; Ikeda, S

    1987-01-01

    The collagenolytic serine proteinase from the pancreas of the catfish (Parasilus asotus) had a pH optimum of 7.5 for native, reconstituted calf skin collagen fibrils. The enzyme was most stable at pH 6-9. The enzyme hydrolyzed heat-denatured collagen (gelatin), casein, hemoglobin and elastin in addition to native collagen but not virtually Tos-Arg-OEe, Bz-Tyr-OEe and Suc-(Ala)3-NA. The enzyme cleaved Leu-Gly (or Gln-Gly), Gly-Ile and Ile-Ala bonds on DNP-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. PMID:3480788

  5. 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

  6. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    PubMed

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  7. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    PubMed

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein. PMID:26795634

  8. Bacterial proteinases as targets for the development of second-generation antibiotics.

    PubMed

    Travis, J; Potempa, J

    2000-03-01

    The emergence of bacterial pathogen resistance to common antibiotics strongly supports the necessity to develop alternative mechanisms for combating drug-resistant forms of these infective organisms. Currently, few pharmaceutical companies have attempted to investigate the possibility of interrupting metabolic pathways other than those that are known to be involved in cell wall biosynthesis. In this review, we describe multiple, novel roles for bacterial proteinases during infection using, as a specific example, the enzymes from the organism Porphyromonas gingivalis, a periodontopathogen, which is known to be involved in the development and progression of periodontal disease. In this manner, we are able to justify the concept of developing synthetic inhibitors against members of this class of enzymes as potential second-generation antibiotics. Such compounds could not only prove valuable in retarding the growth and proliferation of bacterial pathogens but also lead to the use of this class of inhibitors against invasion by other infective organisms. PMID:10708847

  9. The anthelmintic efficacy of natural plant cysteine proteinases against the equine tapeworm, Anoplocephala perfoliata in vitro.

    PubMed

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A E; Behnke, J M

    2016-09-01

    Papaya latex has been demonstrated to be an efficacious anthelmintic against murine, porcine, ovine and canine nematode parasites, and even those infecting poultry, and it has some efficacy against rodent cestodes. The active ingredients of papaya latex are known to be cysteine proteinases (CPs). The experiments described in this paper indicate that CPs in papaya latex, and also those in pineapples, are highly efficacious against the equine cestode Anoplocephala perfoliata in vitro, by causing a significant reduction in motility leading to death of the worms. The susceptibility of A. perfoliata to damage by CPs was considerably greater than that of the rodent cestodes Hymenolepis diminuta and H. microstoma. Our results are the first to report anthelmintic efficacy of CPs against an economically important equine helminth. Moreover, they provide further evidence that the spectrum of activity of CPs is not restricted to nematodes and support the idea that these plant-derived enzymes can be developed into useful broad-spectrum anthelmintics. PMID:26343287

  10. Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides.

    PubMed Central

    Chen, Z X; Potempa, J; Polanowski, A; Renvert, S; Wikström, M; Travis, J

    1991-01-01

    Proteolytic enzymes from the organism Porphyromonas gingivalis are believed to be involved in the development of periodontitis. Studies on both crude extracts and purified trypsinlike enzymes from this organism indicate that substantial stimulation of both amidase and proteinase activities can be obtained during incubation with glycine-containing compounds. We postulate that P. gingivalis may have developed this unusual property to take advantage of the glycine-rich environment which occurs during the periodontitis-associated degradation of gingival collagen. The finding of such a stimulation in crevicular fluids from discrete periodontal sites has been correlated with the presence of P. gingivalis and could be utilized for the early detection of infection by this organism during the onset of periodontitis. PMID:1855999

  11. Protein degradation in Euglena gracilis: Purification and characterization of the major proteinase

    SciTech Connect

    Yoo, Y.J.

    1988-01-01

    Protolysis in a crude extract of Euglena gracilis was characterized by autolysis and the hydrolysis of {sup 125}I-labeled bovine serum albumin ({sup 125}I-BSA). Both procedures showed similar properties: stimulation by dithiothreitol, inhibition by leupeptin, and the same pH optima. Hydrolysis of {sup 125}I-BSA increased with growth stage and with the depletion of nutrient in the medium. The major proteolytic enzyme was purified to near homogeneity from extracts of dark-grown, stationary-phase Euglena gracilis by acid treatment, and by chromatography on CM-cellulose, DEAE-cellulose, Sephadex G-75, and hydroxyapatite using {sup 125}I-BSA as substrate. The molecular weight of the proteinase was 30,000 when determined by gel filtration on Sephadex G-75 and 15,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme therefore appears to be composed of two subunits.

  12. Role of calcium-dependent proteinase in molt-induced claw muscle atrophy

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1984-01-01

    The claw closer muscle of the Bermuda land crab Gecarcinus lateralis undergoes a sequential atrophy and restoration during each intermolt cycle. Muscle protein decreases 40% during proecdysis and is restored following ecdysis. Amino acid incorporation into protein of postecdysial muscle is five times greater than that in anecdysial muscle. Since the rates of protein synthesis in anecdysial and proecdysial muscle are the same it appears that proecdysial muscle atrophy is caused primarily by an increase in protein degradation. A calcium-dependent proteinase (CDP) active at neutral pH has been implicated in the nonlysosomal hydrolysis of myofibrillar proteins. We have examined the role of a CDP in atrophy of the claw closer muscle. The many similarities between crustacean and vertebrate CDPs have established this crustacean system as a simple and convenient model for the role of Ca/sup 2 +/-dependent proteolysis in myofibrillar protein turnover and its manifestation in the structure of the sarcomere. 16 references, 8 figures. (ACR)

  13. Proton transfer pathways in an aspartate-water cluster sampled by a network of discrete states

    NASA Astrophysics Data System (ADS)

    Reidelbach, Marco; Betz, Fridtjof; Mäusle, Raquel Maya; Imhof, Petra

    2016-08-01

    Proton transfer reactions are complex transitions due to the size and flexibility of the hydrogen-bonded networks along which the protons may "hop". The combination of molecular dynamics based sampling of water positions and orientations with direct sampling of proton positions is an efficient way to capture the interplay of these degrees of freedom in a transition network. The energetically most favourable pathway in the proton transfer network computed for an aspartate-water cluster shows the pre-orientation of water molecules and aspartate side chains to be a pre-requisite for the subsequent concerted proton transfer to the product state.

  14. Chemotactic responses of Escherichia coli to small jumps of photoreleased L-aspartate.

    PubMed

    Jasuja, R; Keyoung, J; Reid, G P; Trentham, D R; Khan, S

    1999-03-01

    Computer-assisted motion analysis coupled to flash photolysis of caged chemoeffectors provides a means for time-resolved analysis of bacterial chemotaxis. Escherichia coli taxis toward the amino acid attractant L-aspartate is mediated by the Tar receptor. The physiology of this response, as well as Tar structure and biochemistry, has been studied extensively. The beta-2, 6-dinitrobenzyl ester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acid were synthesized. These compounds liberated L-aspartate and the fluorophore 8-hydroxypyrene 1,3,6-tris-sulfonic acid (pyranine) upon irradiation with near-UV light. Photorelease of the fluorophore was used to define the amplitude and temporal stability of the aspartate jumps employed in chemotaxis experiments. The dependence of chemotactic adaptation times on aspartate concentration, determined in mixing experiments, was best fit by two Tar aspartate-binding sites. Signal processing (excitation) times, amplitudes, and adaptive recovery of responses elicited by aspartate jumps producing less than 20% change in receptor occupancy were characterized in photorelease assays. Aspartate concentration jumps in the nanomolar range elicited measurable responses. The response threshold and sensitivity of swimming bacteria matched those of bacteria tethered to glass by a single flagellum. Stimuli of similar magnitude, delivered either by rapid mixing or photorelease, evoked responses of similar strength, as assessed by recovery time measurements. These times remained proportional to change in receptor occupancy close to threshold, irrespective of prior occupancy. Motor excitation responses decayed exponentially with time. Rates of excitation responses near threshold ranged from 2 to 7 s-1. These values are consistent with control of excitation signaling by decay of phosphorylated pools of the response regulator protein, CheY. Excitation response rates increased slightly with stimulus size

  15. Retrograde transport of (/sup 3/H)-D-aspartate label by cochlear and vestibular efferent neurons

    SciTech Connect

    Schwarz, D.W.; Schwarz, I.E.

    1988-01-01

    (/sup 3/H)-D-aspartic acid was injected into the inner ear of rats. After a six hour survival time, labeled cells were found at all locations known to contain efferent cochlear or vestibular neurons. Most labeled neurons were found in the ipsilateral lateral superior olivary nucleus (LSO), although both ventral nuclei of the trapezoid body (VTB), group E, and the caudal pontine reticular nucleus (CPR) just adjacent to the ascending limb of the facial nerve also contained labeled cells. Because not all efferent neurons in the rat could be previously shown to be cholinergic, aspartate and glutamate are efferent transmitter candidates.

  16. Interaction of the Human Adenovirus Proteinase with Its 11-Amino Acid Cofactor pVIc†

    PubMed Central

    Baniecki, Mary Lynn; McGrath, William J.; McWhirter, Sarah M.; Li, Caroline; Toledo, Diana L.; Pellicena, Patricia; Barnard, Dale L.; Thorn, Kurt S.; Mangel, Walter F.

    2010-01-01

    The interaction of the human adenovirus proteinase (AVP) and AVP–NA complexes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 μM. The binding of AVP to 12-mer single-stranded DNA decreased the Kd for the binding of pVIc to AVP to 0.09 μM. The pVIc–AVP complex hydrolyzed the substrate with a Michaelis constant (Km) of 3.7 μM and a catalytic rate constant (kcat) of 1.1 s−1 In the presence of DNA, the Km increased less than 2-fold, and the kcat increased 3-fold. Alanine-scanning mutagenesis was performed to determine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Two amino acid residues, Gly1′ and Phe11′, were the major determinants in the binding of pVIc to AVP, while Val2′ and Phe11′ were the major determinants in stimulating enzyme activity. Binding of AVP to DNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP. Binding of either or both of the cofactors, pVIc or the viral DNA, to AVP did not dramatically alter its secondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably by prematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly, thereby aborting the infection. PMID:11591154

  17. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

    PubMed

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-08-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  18. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

    PubMed Central

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-01-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [3H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4+ lymphocyte proliferation but did not affect the proliferation of CD8+ cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  19. Secretion of mucus proteinase inhibitor and elafin by Clara cell and type II pneumocyte cell lines.

    PubMed

    Sallenave, J M; Silva, A; Marsden, M E; Ryle, A P

    1993-02-01

    The regulation of proteinases secreted by neutrophils is very important for the prevention of tissue injury. We recently described the isolation of elafin from bronchial secretions, a new elastase-specific inhibitor that is also found in the skin of patients with psoriasis. In this study, we investigated the secretion of elafin and mucus proteinase inhibitor (MPI), another inhibitor showing sequence similarity with elafin, in two lung carcinoma cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. The results presented show that the two inhibitors are produced when the cells are cultured either in serum-free or in serum-containing media. MPI was detected immunologically as a unique molecule of M(r) 14 kD, in accordance with previous studies. Conversely, one or two elafin-immunoreactive species were detected depending on the cell line: a 12- to 14-kD species was observed in the A549 cell line, regardless of the culture conditions, whereas in the NCI-H322 cell line we detected a 6-kD species in serum-containing (10% fetal calf serum) conditions and a 12- to 14-kD species in serum-free conditions. The 12- to 14-kD molecule probably represents an active precursor of elafin. Whether the cleavage of the 12- to 14-kD precursor giving rise to the elafin molecule is of any physiologic significance is not known. In showing for the first time that MPI and elafin (and its precursor) are secreted by the A549 cell line, this report implicates the type II alveolar cell in the defense of the peripheral lung against the neutrophil elastase secreted during inflammation. PMID:8427705

  20. Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3.

    PubMed

    Piesche, Matthias; Hildebrandt, York; Chapuy, Björn; Wulf, Gerald G; Trümper, Lorenz; Schroers, Roland

    2009-07-23

    Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegener's granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recognize antigens in the context of HLA-class-II-molecules, identification of HLA-class-II-associated peptide-epitopes from self-antigens such as PRTN3 is required. Here, we analyzed T-cell responses against proteinase 3 using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. We first screened a panel of ten epitope peptide candidates selected with the TEPITOPE program and found that nine out of ten peptides induced PRTN3 peptide-specific proliferation of T-cells with precursor frequencies of 0-1.1 x 10(-6). For one peptide-epitope, PRTN3(235), T-cell-clones were demonstrated to be capable of recognizing naturally processed protein antigen in a HLA-DR-restricted fashion. PRTN3(235)-specific T-cells could be stimulated from the blood of healthy individuals with multiple HLA-DR-genotypes. In summary, the identified PRTN3(235)-epitope can be used to study the role of CD4+ TH- and Treg-cells in immune responses against PRTN3 in leukemia patients and patients with Wegener's disease. PMID:19446593

  1. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    PubMed

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years. PMID:27194832

  2. Antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera).

    PubMed

    Kim, Jae Hak; Mullin, Christopher A

    2003-04-01

    Low-molecular-weight peptidyl proteinase inhibitors (PIs) including leupeptin, calpain inhibitor I, and calpeptin were found to be potent antifeedants for adult western corn rootworm (WCR) against the phagostimulation of cucurbitacin B (Cuc B) or a corn pollen extract (CPE). Leupeptin was the strongest (ED50 = 0.36 and 0.55 nmol/disk for Cuc B and CPE, respectively) among PIs tested with an antifeedant potency much stronger than the steroid progesterone (ED50 = 2.29 and 5.05 nmol/disk for Cuc B and CPE, respectively), but slightly less than the reference alkaloid, strychnine (ED50 = 0.17 and 0.37 nmol/disk for Cuc B and CPE, respectively). All active PIs contain a di- or tripeptidyl aldehyde moiety, indicating that PIs exert their antifeedant effects by covalent interaction with putative sulfhydryl (SH) groups on taste receptors as do these PIs with cysteine proteinases. However, opposite inhibition potency against Cuc B versus CPE by two thiol-group reducing agents, DTT and L-cysteine, and the results with other cysteine-modifying reagents obscure the net functional role of SH groups at WCR taste chemoreceptors. Surprisingly, the model phagostimulant for diabroticites, Cuc B, was more easily counteracted by these feeding deterrents than the stimulants present in CPE. Three-dimensional structure-antifeedant relationships for the PIs suggest that a novel taste chemoreception mechanism exists for these peptidyl aldehydes or that they fit partially into a strychnine binding pocket on protein chemoreceptors. Favorable economic benefit may be achieved if PIs are discovered to be useful in adult WCR control, since both pre- and postingestive sites would be targeted. PMID:12775144

  3. Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen

    PubMed Central

    Reiners, Katrin S; Hansen, Hinrich P; Krüssmann, Anne; Schön, Gisela; Csernok, Elena; Gross, Wolfgang L; Engert, Andreas; von Strandmann, Elke Pogge

    2004-01-01

    Wegener's granulomatosis (WG) is a rare disease characterized by granulomatous lesions, small vessel vasculitis and the presence of anti-neutrophil cytoplasmic autoantibodies (C-ANCAs) in the sera of affected patients. Their main target antigen is proteinase 3 (PR3), a neutrophil and monocyte-derived neutral serine protease. Since the standard treatment of this severe autoimmune disease, with cyclophosphamide and corticosteroids, is associated with potential side-effects, the development of a more specific immunotherapeutic agent is warranted. The key role of ANCA in the pathogenesis of vasculitis and the effectiveness of anti-CD20 antibodies in patients with refractory WG points towards the importance of B cells in WG. We thus evaluated a new approach to selectively eliminate PR3-specific autoreactive B cells by targeting the B-cell receptor. For this purpose we used a bifunctional recombinant fusion protein consisting of the antigen PR3 and a toxin. The cytotoxic component of this novel fusion protein was the ribonuclease angiogenin, a human toxin with low immunogenicity. The toxin was stabilized by exchanging the catalytically relevant histidine in position 44 with glutamine to eliminate the autoproteolytic activity. PR3H44Q was fused either to the N terminus or to the C terminus of angiogenin. The recombinant proteins were expressed in 293T cells. Binding assays demonstrated the appropriate size and recognition by anti-PR3 antibodies. Using TUNEL technology, we demonstrated that these autoantigen toxins kill proteinase 3-specific B-cell hybridomas selectively by inducing apoptosis. The data indicate that autoantigen-toxins are promising tools in the treatment or co-treatment of autoimmune diseases in which the antigen is known. PMID:15147566

  4. High order generalized permutational fractional Fourier transforms

    NASA Astrophysics Data System (ADS)

    Ran, Qi-Wen; Yuan, Lin; Tan, Li-Ying; Ma, Jing; Wang, Qi

    2004-02-01

    We generalize the definition of the fractional Fourier transform (FRFT) by extending the new definition proposed by Shih. The generalized FRFT, called the high order generalized permutational fractional Fourier transform (HGPFRFT), is a generalized permutational transform. It is shown to have arbitrary natural number M periodic eigenvalues not only with respect to the order of Hermite-Gaussian functions but also to the order of the transform. This HGPFRFT will be reduced to the original FRFT proposed by Namias and Liu's generalized FRFT and Shih's FRFT at the three limits with M = +infty, M = 4k (k is a natural number) and M = 4, respectively. Therefore the HGPFRFT introduces a comprehensive approach to Shih's FRFT and the original definition. Some important properties of HGPFRFT are discussed. Lastly the results of computer simulations and symbolic representations of the transform are provided.

  5. Electro-optic Imaging Fourier Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin

    2005-01-01

    JPL is developing an innovative compact, low mass, Electro-Optic Imaging Fourier Transform Spectrometer (E-O IFTS) for hyperspectral imaging applications. The spectral region of this spectrometer will be 1 - 2.5 micron (1000-4000/cm) to allow high-resolution, high-speed hyperspectral imaging applications. One application will be the remote sensing of the measurement of a large number of different atmospheric gases simultaneously in the same airmass. Due to the use of a combination of birefringent phase retarders and multiple achromatic phase switches to achieve phase delay, this spectrometer is capable of hyperspectral measurements similar to that of the conventional Fourier transform spectrometer but without any moving parts. In this paper, the principle of operations, system architecture and recent experimental progress will be presented.

  6. Electro-optic Imaging Fourier Transform Spectrometer

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin

    2005-01-01

    JPL is developing an innovative compact, low mass, Electro-Optic Imaging Fourier Transform Spectrometer (E-0IFTS) for hyperspectral imaging applications. The spectral region of this spectrometer will be 1 - 2.5 pm (1000 -4000 cm-') to allow high-resolution, high-speed hyperspectral imaging applications [l-51. One application will be theremote sensing of the measurement of a large number of different atmospheric gases simultaneously in the sameairmass. Due to the use of a combination of birefiingent phase retarders and multiple achromatic phase switches toachieve phase delay, this spectrometer is capable of hyperspectral measurements similar to that of the conventionalFourier transform spectrometer but without any moving parts. In this paper, the principle of operations, systemarchitecture and recent experimental progress will be presen.

  7. Miniaturization of holographic Fourier-transform spectrometers.

    PubMed

    Agladze, Nikolay I; Sievers, Albert J

    2004-12-20

    Wave propagation equations in the stationary-phase approximation have been used to identify the theoretical bounds of a miniature holographic Fourier-transform spectrometer (HFTS). It is demonstrated that the HFTS throughput can be larger than for a scanning Fourier-transform spectrometer. Given room- or a higher-temperature constraint, a small HFTS has the potential to outperform a small multichannel dispersive spectrograph with the same resolving power because of the size dependence of the signal-to-noise ratio. These predictions are used to analyze the performance of a miniature HFTS made from simple optical components covering a broad spectral range from the UV to the near IR. The importance of specific primary aberrations in limiting the HFTS performance has been both identified and verified. PMID:15646777

  8. Fourier analysis: from cloaking to imaging

    NASA Astrophysics Data System (ADS)

    Wu, Kedi; Cheng, Qiluan; Wang, Guo Ping

    2016-04-01

    Regarding invisibility cloaks as an optical imaging system, we present a Fourier approach to analytically unify both Pendry cloaks and complementary media-based invisibility cloaks into one kind of cloak. By synthesizing different transfer functions, we can construct different devices to realize a series of interesting functions such as hiding objects (events), creating illusions, and performing perfect imaging. In this article, we give a brief review on recent works of applying Fourier approach to analysis invisibility cloaks and optical imaging through scattering layers. We show that, to construct devices to conceal an object, no constructive materials with extreme properties are required, making most, if not all, of the above functions realizable by using naturally occurring materials. As instances, we experimentally verify a method of directionally hiding distant objects and create illusions by using all-dielectric materials, and further demonstrate a non-invasive method of imaging objects completely hidden by scattering layers.

  9. Fourier spectroscopy on planetary missions including Voyager

    NASA Technical Reports Server (NTRS)

    Hanel, R. A.

    1981-01-01

    In the last dozen years spaceborne Fourier transform spectrometers have obtained infrared emission spectra of Earth, Mars, Jupiter, Saturn and Titan as well as of the Galilean and other Saturnian satellites and Saturn's rings. Intercomparisons of the properties of planetary atmospheres and of the characteristics of solid surfaces are now feasible. The principles of remotely sensing the environment on a planetary body are dicussed. Special consideration is given to the most recent results obtained by the Voyager infrared investigation on the Saturn system.

  10. Exponential Approximations Using Fourier Series Partial Sums

    NASA Technical Reports Server (NTRS)

    Banerjee, Nana S.; Geer, James F.

    1997-01-01

    The problem of accurately reconstructing a piece-wise smooth, 2(pi)-periodic function f and its first few derivatives, given only a truncated Fourier series representation of f, is studied and solved. The reconstruction process is divided into two steps. In the first step, the first 2N + 1 Fourier coefficients of f are used to approximate the locations and magnitudes of the discontinuities in f and its first M derivatives. This is accomplished by first finding initial estimates of these quantities based on certain properties of Gibbs phenomenon, and then refining these estimates by fitting the asymptotic form of the Fourier coefficients to the given coefficients using a least-squares approach. It is conjectured that the locations of the singularities are approximated to within O(N(sup -M-2), and the associated jump of the k(sup th) derivative of f is approximated to within O(N(sup -M-l+k), as N approaches infinity, and the method is robust. These estimates are then used with a class of singular basis functions, which have certain 'built-in' singularities, to construct a new sequence of approximations to f. Each of these new approximations is the sum of a piecewise smooth function and a new Fourier series partial sum. When N is proportional to M, it is shown that these new approximations, and their derivatives, converge exponentially in the maximum norm to f, and its corresponding derivatives, except in the union of a finite number of small open intervals containing the points of singularity of f. The total measure of these intervals decreases exponentially to zero as M approaches infinity. The technique is illustrated with several examples.

  11. Fourier analysis of the SOR iteration

    NASA Technical Reports Server (NTRS)

    Leveque, R. J.; Trefethen, L. N.

    1986-01-01

    The SOR iteration for solving linear systems of equations depends upon an overrelaxation factor omega. It is shown that for the standard model problem of Poisson's equation on a rectangle, the optimal omega and corresponding convergence rate can be rigorously obtained by Fourier analysis. The trick is to tilt the space-time grid so that the SOR stencil becomes symmetrical. The tilted grid also gives insight into the relation between convergence rates of several variants.

  12. Electrocardiogram analysis through time discrete Fourier transform

    NASA Astrophysics Data System (ADS)

    Lancaster, Cameron; Zhang, Guoping

    2011-03-01

    The motivation for this research is to find an alternative method to diagnose heart conditions. This can be accomplished by analyzing wave patterns in EKG data, and using the Fourier Transform to compare steady wave patterns against fibrillating wave patterns. The two main contributors are the following: heart electricity and Fourier Transform. Also, it is recognized that ion movement has potential to change the frequency in any heart beat signal. This effect is caused due to a strong electrostatic attraction that causes the membrane capacitance to build charge. For a single ion focus, the Nernst Potential influences the equilibrium potential for the membrane of an ion. If two or more ions are contributing to an electric field charge, the Goldman-Hodgkin-Katz will find the membrane equilibrium potential. If a membrane has an efflux, or influx, of ions, then it is possible to get the passive flow of the electric current to zero. In continued research, we will gain knowledge of solving equations; such as ionic flux, quantitative diffusion, electric current density, and more. The finishing portion of this research will be to compare the Fourier Transformed wave graphs to determine heart conditions. Supported by U.S. Department of Energy under Contract No. DE-FG02-06ER46304 and Indiana State University.

  13. Coherent Fourier transform electrical pulse shaping.

    PubMed

    Xiao, Shijun; Weiner, Andrew M

    2006-04-01

    Fourier synthesis pulse shaping methods allowing generation of programmable, user defined femtosecond optical waveforms have been widely applied in ultrafast optical science and technology. In the electrical domain, arbitrary waveform generation is well established at frequencies below approximately 1 GHz, but is difficult at higher frequencies due to limitations in digital-to-analog converter technology. In this paper we demonstrate a method for electrical waveform synthesis at substantially higher frequencies (approximately 20 GHz electrical bandwidth) by combining Fourier optical pulse shaping (extended to hyperfine frequency resolution) and heterodyne optical to electrical conversion. Our scheme relies on coherent manipulation of fields and phases at all stages, both for processing in the optical domain and for conversion from the optical to the electrical domain. We illustrate this technique through a number of examples, including programmable retardation or advancement of short electrical pulses in time over a range exceeding ten pulse durations. Such optically implemented, coherent Fourier transform electrical pulse shaping should open new prospects in ultrawideband electromagnetics. PMID:19516448

  14. Fourier analysis of quadratic phase interferograms

    NASA Astrophysics Data System (ADS)

    Muñoz-Maciel, Jesús; Mora-González, Miguel; Casillas-Rodríguez, Francisco J.; Peña-Lecona, Francisco G.

    2015-06-01

    A phase demodulation method from a single interferogram with a quadratic phase term is developed. The fringe pattern being analysed may contain circular, elliptic or astigmatic fringes. The Fourier transform of such interferograms is seen to be also a sine or a cosine of a second order polynomial in both the real and imaginary parts. In this work we take a discrete Fourier transform of the fringe patterns and then we take separate inverse discrete transforms of the real and imaginary parts of the frequency spectrum. This results in two new interferograms corresponding to the sine and cosine of the quadratic term of the phase modulated by the sine and cosine of the linear term. The linear term of these interferograms may be recovered with similar procedures of fringe analysis from open fringe interferograms. Once the linear term is retrieved the quadratic phase of the interferogram being analysed can also be calculated. The present approach is also being investigated for interferograms with nearly circularly symmetry given that the phase contains some tilt. The described procedure of Fourier analysis from quadratic phase interferograms of nearly symmetric interferograms could be used instead of complex and time consuming algorithms for phase recovery from fringe patterns with closed fringes. Finally, the method is tested in simulated and real data.

  15. Novel Role for Proteinase-activated Receptor 2 (PAR2) in Membrane Trafficking of Proteinase-activated Receptor 4 (PAR4)*

    PubMed Central

    Cunningham, Margaret R.; McIntosh, Kathryn A.; Pediani, John D.; Robben, Joris; Cooke, Alexandra E.; Nilsson, Mary; Gould, Gwyn W.; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

    2012-01-01

    Proteinase-activated receptors 4 (PAR4) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR4 remain unknown. Here, we report novel features of the intracellular trafficking of PAR4 to the plasma membrane. PAR4 was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR4 protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R183AR → A183AA), mutation of which allowed efficient membrane delivery of PAR4. Interestingly, co-expression with PAR2 facilitated plasma membrane delivery of PAR4, an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR2 and PAR4. PAR2 also enhanced glycosylation of PAR4 and activation of PAR4 signaling. Our results identify a novel regulatory role for PAR2 in the anterograde traffic of PAR4. PAR2 was shown to both facilitate and abrogate protein interactions with PAR4, impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR4 in normal physiology and disease. PMID:22411985

  16. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

    PubMed

    Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

    2012-05-11

    Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease. PMID:22411985

  17. Fourier analysis of biological signals with a programmable calculator.

    PubMed

    Avolio, A P

    1984-01-01

    A Fourier analysis program written for the Texas Instruments TI-59 programmable calculator is presented. The program determines the Fourier coefficients of any time varying periodic signal. PMID:6546911

  18. Fourier removal of stripe artifacts in IRAS images

    NASA Technical Reports Server (NTRS)

    Van Buren, Dave

    1987-01-01

    By working in the Fourier plane, approximate removal of stripe artifacts in IRAS images can be effected. The image of interest is smoothed and subtracted from the original, giving the high-spatial-frequency part. This 'filtered' image is then clipped to remove point sources and then Fourier transformed. Subtracting the Fourier components contributing to the stripes in this image from the Fourier transform of the original and transforming back to the image plane yields substantial removal of the stripes.

  19. Cloning and expression of genes of aspartate-family amino acid aiosynthesis from medicago truncatula

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four of the amino acids that must be acquired in the human diet, lysine, threonine, methionine and isoleucine, are derived from a common precursor, aspartate, and are produced in a branched, highly-regulated, biosynthetic pathway. Moreover, the common dietary sources of plant proteins, cereals grain...

  20. Threonine Overproduction in Transgenic Tobacco Plants Expressing a Mutant Desensitized Aspartate Kinase of Escherichia coli1

    PubMed Central

    Shaul, Orit; Galili, Gad

    1992-01-01

    In higher plants, the synthesis of the essential amino acid threonine is regulated primarily by the sensitivity of the first enzyme in its biosynthetic pathway, aspartate kinase, to feedback inhibition by threonine and lysine. We aimed to study the potential of increasing threonine accumulation in plants by means of genetic engineering. This was addressed by the expression of a mutant, desensitized aspartate kinase derived from Escherichia coli either in the cytoplasm or in the chloroplasts of transgenic tobacco (Nicotiana Tabacum cv Samsun NN) plants. Both types of transgenic plants exhibited a significant overproduction of free threonine. However, threonine accumulation was higher in plants expressing the bacterial enzyme in the chloroplast, indicating that compartmentalization of aspartate kinase within this organelle was important, although not essential. Threonine overproduction in leaves was positively correlated with the level of the desensitized enzyme. Transgenic plants expressing the highest leaf aspartate kinase activity also exhibited a slight increase in the levels of free lysine and isoleucine, both of which share a common biosynthetic pathway with threonine, but showed no significant change in the level of other free amino acids. The present study proposes a new molecular biological approach to increase the limiting content of threonine in higher plants. PMID:16653099

  1. Genome-wide identification, evolutuionary and expression analysis of aspartic proteases gene superfamily in grape

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartic proteases (APs) are a large family of proteolytic enzymes in vertebrates, plants, yeast, nematodes, parasites, fungi, and viruses. In plants, they are involved in many biological processes, such as plant senescence, stress response, programmed cell death, and reproduction. Prior to the pr...

  2. A Green Polymerization of Aspartic Acid for the Undergraduate Organic Laboratory

    ERIC Educational Resources Information Center

    Bennett, George D.

    2005-01-01

    The green polymerization of aspartic acid carried out during an organic-inorganic synthesis laboratory course for undergraduate students is described. The procedure is based on work by Donlar Corporation, a Peru, Illinois-based company that won a Green Chemistry Challenge Award in 1996 in the Small Business category for preparing thermal…

  3. Identification of a small molecule [beta]-secretase inhibitor that binds without catalytic aspartate engagement

    SciTech Connect

    Steele, Thomas G.; Hills, Ivory D.; Nomland, Ashley A.; de León, Pablo; Allison, Timothy; McGaughey, Georgia; Colussi, Dennis; Tugusheva, Katherine; Haugabook, Sharie J.; Espeseth, Amy S.; Zuck, Paul; Graham, Samuel L.; Stachel, Shawn J.

    2010-09-02

    A small molecule inhibitor of beta-secretase with a unique binding mode has been developed. Crystallographic determination of the enzyme-inhibitor complex shows the catalytic aspartate residues in the active site are not engaged in inhibitor binding. This unprecedented binding mode in the field of aspartyl protease inhibition is described.

  4. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  5. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  6. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  8. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  9. Pneumocystis Jirovecii Pneumonia in a Patient with Anti-N-Methyl-D-Aspartate Receptor Postherpetic Encephalitis.

    PubMed

    García-Moreno, Jorge; Igartua Laraudogoitia, Jon; Montes Ros, Milagrosa

    2016-07-01

    Anti-N-methyl-D-aspartate receptor encephalitis is a neuroimmunologic disorder that has been increasingly diagnosed during the past 5 years. It provokes a predictable syndrome treated with several immunomodulatory agents, such as corticosteroids and/or biologics. We managed a child with this disease who developed Pneumocystis jirovecii pneumonia as a direct infectious complication of the use of rituximab. PMID:27093160

  10. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile. PMID:27437081

  11. Some Applications of Fourier's Great Discovery for Beginners

    ERIC Educational Resources Information Center

    Kraftmakher, Yaakov

    2012-01-01

    Nearly two centuries ago, Fourier discovered that any periodic function of period T can be presented as a sum of sine waveforms of frequencies equal to an integer times the fundamental frequency [omega] = 2[pi]/T (Fourier's series). It is impossible to overestimate the importance of Fourier's discovery, and all physics or engineering students…

  12. Fourier Transforms Simplified: Computing an Infrared Spectrum from an Interferogram

    ERIC Educational Resources Information Center

    Hanley, Quentin S.

    2012-01-01

    Fourier transforms are used widely in chemistry and allied sciences. Examples include infrared, nuclear magnetic resonance, and mass spectroscopies. A thorough understanding of Fourier methods assists the understanding of microscopy, X-ray diffraction, and diffraction gratings. The theory of Fourier transforms has been presented in this "Journal",…

  13. Optical image encryption based on multifractional Fourier transforms.

    PubMed

    Zhu, B; Liu, S; Ran, Q

    2000-08-15

    We propose a new image encryption algorithm based on a generalized fractional Fourier transform, to which we refer as a multifractional Fourier transform. We encrypt the input image simply by performing the multifractional Fourier transform with two keys. Numerical simulation results are given to verify the algorithm, and an optical implementation setup is also suggested. PMID:18066153

  14. Insights into the behaviour of biomolecules on the early Earth: The concentration of aspartate by layered double hydroxide minerals

    NASA Astrophysics Data System (ADS)

    Grégoire, Brian; Erastova, Valentina; Geatches, Dawn L.; Clark, Stewart J.; Greenwell, H. Christopher; Fraser, Donald G.

    2016-03-01

    The role of mineral surfaces in concentrating and facilitating the polymerisation of simple protobiomolecules during the Hadean and Archean has been the subject of much research in order to constrain the conditions that may have led to the origin of life on early Earth. Here we examine the adsorption of the amino acid aspartate on layered double hydroxide minerals, and use a combined computer simulation - experimental spectroscopy approach to gain insight into the resulting structures of the host-aspartate material. We show that the uptake of aspartate occurs in alkaline solution by anion exchange of the dianion form of aspartate, rather than by surface adsorption. Anion exchange only occurs at values of pH where a significant population of aspartate has the amino group deprotonated, and is then highly efficient up to the mineral anion exchange capacity.

  15. DNA interaction with octahedral and square planar Ni(II) complexes of aspartic-acid Schiff-bases

    NASA Astrophysics Data System (ADS)

    Sallam, S. A.; Orabi, A. S.; Abbas, A. M.

    2011-12-01

    Ni(II) complexes of (S,E)-2-(2-OHbenzilydene)aspartic acid; (S,E)-2-(2,3-diOHbenzilydene)aspartic acid-; (S,E)-2-(2,4-diOH-benzilydene)aspartic acid; (S,E)-2-(2,5-diOHbenzilydene)aspartic acid and (S,E)-2-((2-OHnaphthalene-1-yl)methylene)aspartic acid Schiff-bases have been synthesized by template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1H nmr spectra as well as thermal analysis (TG, DTG, DTA). The Schiff-bases are dibasic tridentate or tetradentate donors and the complexes have square planar and octahedral structures. The complexes decompose in two or three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy.

  16. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    PubMed

    Onstott, T C; Magnabosco, C; Aubrey, A D; Burton, A S; Dworkin, J P; Elsila, J E; Grunsfeld, S; Cao, B H; Hein, J E; Glavin, D P; Kieft, T L; Silver, B J; Phelps, T J; van Heerden, E; Opperman, D J; Bada, J L

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples. PMID:24289240

  17. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    SciTech Connect

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  18. Taurine attenuates D-[3H]aspartate release evoked by depolarization in ischemic corticostriatal slices.

    PubMed

    Molchanova, Svetlana M; Oja, Simo S; Saransaari, Pirjo

    2006-07-12

    Taurine is thought to be protective in ischemia due to its neuroinhibitory effects. The present aim was to assess the ability of taurine to attenuate glutamate release evoked by ischemia and to determine which component of this release is affected. The release of preloaded D-[(3)H]aspartate (a non-metabolized analog of glutamate) from superfused murine corticostriatal slices was used as index of glutamate release. Preincubation of corticostriatal slices with 10 mM taurine reduced the D-[(3)H]aspartate release evoked by either chemical ischemia (0.5 mM NaCN in glucose-free medium) or oxygen-glucose deprivation. The taurine uptake inhibitor guanidinoethanesulfonate (5 mM), the glycine receptor antagonist strychnine (0.1 mM) and the GABA(A) receptor antagonist bicuculline (0.1 mM) did not block the taurine effect. To determine which component of ischemia-induced glutamate release is affected by taurine, three pathways of this release were pharmacologically modeled. Unlabeled D-aspartate (0.5 mM) and hypo-osmotic medium (NaCl reduced by 50 mM) evoked D-[(3)H]aspartate release via homoexchange and hypo-osmotic release pathways, respectively. Taurine did not influence these pathways. However, it suppressed the synaptic release of D-[(3)H]aspartate evoked by the voltage-gated sodium channel opener veratridine (0.1 mM). Taurine thus reduces glutamate release under ischemic conditions by affecting the depolarization-evoked component. PMID:16781687

  19. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    SciTech Connect

    Onstott, T. C.; Aubrey, A.D.; Kieft, T L; Silver, B J; Phelps, Tommy Joe; Van Heerden, E.; Opperman, D. J.; Bada, J L.

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 C and 1 2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  20. Does Aspartic Acid Racemization Constrain the Depth Limit of the Subsurface Biosphere?

    NASA Technical Reports Server (NTRS)

    Onstott, T C.; Magnabosco, C.; Aubrey, A. D.; Burton, A. S.; Dworkin, J. P.; Elsila, J. E.; Grunsfeld, S.; Cao, B. H.; Hein, J. E.; Glavin, D. P.; Kieft, T. L.; Silver, B. J.; Phelps, T. J.; Heerden, E. Van; Opperman, D. J.; Bada, J. L.

    2013-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of approximately 89 years for 1 km depth and 27 C and 1-2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  1. Insulin degludec and insulin aspart: novel insulins for the management of diabetes mellitus

    PubMed Central

    Atkin, Stephen; Javed, Zeeshan; Fulcher, Gregory

    2015-01-01

    Patients with type 2 diabetes mellitus require insulin as disease progresses to attain or maintain glycaemic targets. Basal insulin is commonly prescribed initially, alone or with one or more rapid-acting prandial insulin doses, to limit mealtime glucose excursions (a basal–bolus regimen). Both patients and physicians must balance the advantages of improved glycaemic control with the risk of hypoglycaemia and increasing regimen complexity. The rapid-acting insulin analogues (insulin aspart, insulin lispro and insulin glulisine) all have similar pharmacokinetic and pharmacodynamic characteristics and clinical efficacy/safety profiles. However, there are important differences in the pharmacokinetic and pharmacodynamic profiles of basal insulins (insulin glargine, insulin detemir and insulin degludec). Insulin degludec is an ultra-long-acting insulin analogue with a flat and stable glucose-lowering profile, a duration of action exceeding 30 h and less inter-patient variation in glucose-lowering effect than insulin glargine. In particular, the chemical properties of insulin degludec have allowed the development of a soluble co-formulation with prandial insulin aspart (insulin degludec/insulin aspart) that provides basal insulin coverage for at least 24 h with additional mealtime insulin for one or two meals depending on dose frequency. Pharmacokinetic and pharmacodynamic studies have shown that the distinct, long basal glucose-lowering action of insulin degludec and the prandial glucose-lowering effect of insulin aspart are maintained in the co-formulation. Evidence from pivotal phase III clinical trials indicates that insulin degludec/insulin aspart translate into sustained glycaemic control with less hypoglycaemia and the potential for a simpler insulin regimen with fewer daily injections. PMID:26568812

  2. Insulin degludec and insulin aspart: novel insulins for the management of diabetes mellitus.

    PubMed

    Atkin, Stephen; Javed, Zeeshan; Fulcher, Gregory

    2015-11-01

    Patients with type 2 diabetes mellitus require insulin as disease progresses to attain or maintain glycaemic targets. Basal insulin is commonly prescribed initially, alone or with one or more rapid-acting prandial insulin doses, to limit mealtime glucose excursions (a basal-bolus regimen). Both patients and physicians must balance the advantages of improved glycaemic control with the risk of hypoglycaemia and increasing regimen complexity. The rapid-acting insulin analogues (insulin aspart, insulin lispro and insulin glulisine) all have similar pharmacokinetic and pharmacodynamic characteristics and clinical efficacy/safety profiles. However, there are important differences in the pharmacokinetic and pharmacodynamic profiles of basal insulins (insulin glargine, insulin detemir and insulin degludec). Insulin degludec is an ultra-long-acting insulin analogue with a flat and stable glucose-lowering profile, a duration of action exceeding 30 h and less inter-patient variation in glucose-lowering effect than insulin glargine. In particular, the chemical properties of insulin degludec have allowed the development of a soluble co-formulation with prandial insulin aspart (insulin degludec/insulin aspart) that provides basal insulin coverage for at least 24 h with additional mealtime insulin for one or two meals depending on dose frequency. Pharmacokinetic and pharmacodynamic studies have shown that the distinct, long basal glucose-lowering action of insulin degludec and the prandial glucose-lowering effect of insulin aspart are maintained in the co-formulation. Evidence from pivotal phase III clinical trials indicates that insulin degludec/insulin aspart translate into sustained glycaemic control with less hypoglycaemia and the potential for a simpler insulin regimen with fewer daily injections. PMID:26568812

  3. Effects of data gaps on Fourier Analysis

    NASA Astrophysics Data System (ADS)

    Negrea, Catalin; Munteanu, Costel; Echim, Marius

    2014-05-01

    Fourier Analysis is a vital and widely used tool in all branches of science that require advanced data processing. The method is often used via the Fast Fourier Transform (FFT) implementation readily available in most programming languages. This is a valid approach for data sets with equally spaced data points and no gaps. Such conditions are not always met in real situations where corrections and adjustments to the method are needed. We investigate the intrinsic limitations of four such methods when data gaps are present: 1) linear interpolations and FFT, 2) a direct implementation of the Discrete Fourier Transform, 3) a Z-Transform and 4) the Lomb-Scargle algorithm. Theoretical analysis tools can provide an insight as to the likely problems of such methods and we discuss the likely modifications to the computed spectra. Also, a time series with no data gaps and a constant sampling frequency is altered by introducing several gap configurations and the resulting spectra with the four methods are compared to highlight changes with respect to the original spectrum. Effects on the amplitude and phase of the resulting power spectral densities are analyzed for non-uniformly sampled solar wind data provided by the Venus Express spacecraft. Phase effects are also studied in the context of a sliding window approach. Research supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 313038/STORM, and a grant of the Romanian Ministry of National Education, CNCS - UEFISCDI, project number PN-II-ID-PCE-2012-4-0418.

  4. CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia.

    PubMed

    Scheibenbogen, Carmen; Letsch, Anne; Thiel, Eckhard; Schmittel, Alexander; Mailaender, Volker; Baerwolf, Steffi; Nagorsen, Dirk; Keilholz, Ulrich

    2002-09-15

    Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2-positive AML patients by interferon-gamma (IFN-gamma) ELISPOT assay and flow cytometry for intracellular IFN-gamma and in 3 additional patients by flow cytometry only. T cells producing IFN-gamma in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3(+)CD8(+)CD45RA(+) CCR7(-) T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B(+). These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines. PMID:12200377

  5. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

    PubMed Central

    Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

    1997-01-01

    Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

  6. A STUDY OF THE INHIBITION OF STREPTOCOCCAL PROTEINASE BY SERA OF NORMAL AND IMMUNE ANIMALS AND OF PATIENTS INFECTED WITH GROUP A HEMOLYTIC STREPTOCOCCI

    PubMed Central

    Todd, Edgar W.

    1947-01-01

    Antiproteinase sera were prepared by immunizing horses with filtrates from a selected strain of group A streptococcus. This strain, which produced high titred proteinase but no erythrogenic toxin, was selected from forty-two strains of group A streptococci which produced varying amounts of proteinase. A few strains belonging to groups B, C, and G were also tested; they were all proteinase-negative. Methods are described for titrating streptococcal proteinase in crude culture filtrates and for measuring the antiproteinase activity of serum. The antiproteinase titres of sera from immunized horses ranged from 125 units to 1,000 units per cc. in contrast to the low titres of normal horse sera, only 5 per cent of which had titres as high as 10 to 30 units per cc. The available evidence suggests that the antiproteinase activity of immune sera is dependent on the action of specific antibody for streptococcal proteinase. Patients infected with group A streptococci do not develop high anti-proteinase titres. There appears to be no correlation between the occurrence of rheumatic fever and the antiproteinase titre of the patient's serum. PMID:19871638

  7. Secret sharing based on quantum Fourier transform

    NASA Astrophysics Data System (ADS)

    Yang, Wei; Huang, Liusheng; Shi, Runhua; He, Libao

    2013-07-01

    Secret sharing plays a fundamental role in both secure multi-party computation and modern cryptography. We present a new quantum secret sharing scheme based on quantum Fourier transform. This scheme enjoys the property that each share of a secret is disguised with true randomness, rather than classical pseudorandomness. Moreover, under the only assumption that a top priority for all participants (secret sharers and recovers) is to obtain the right result, our scheme is able to achieve provable security against a computationally unbounded attacker.

  8. Phase amplitude conformal symmetry in Fourier transforms

    NASA Astrophysics Data System (ADS)

    Kuwata, S.

    2015-04-01

    For the Fourier transform ℑ : L2(R) → L2(R) of a complex-valued even or odd function ψ, it is found that the amplitude invariance |ℑψ| = |ψ| leads to a phase invariance or inversion as arg(ℑψ) = ±argψ + θ (θ = constant). The converse holds unless arg ψ = constant. The condition |ψ| = |ℑψ| is required in dealing with, for example, the minimum uncertainty relation between position and momentum. Without the evenness or oddness of ψ, |ℑψ| = |ψ| does not necessarily imply arg(ℑψ) = ±argψ + θ, nor is the converse.

  9. Noise figure of amplified dispersive Fourier transformation

    SciTech Connect

    Goda, Keisuke; Jalali, Bahram

    2010-09-15

    Amplified dispersive Fourier transformation (ADFT) is a powerful tool for fast real-time spectroscopy as it overcomes the limitations of traditional optical spectrometers. ADFT maps the spectrum of an optical pulse into a temporal waveform using group-velocity dispersion and simultaneously amplifies it in the optical domain. It greatly simplifies spectroscopy by replacing the diffraction grating and detector array in the conventional spectrometer with a dispersive fiber and single-pixel photodetector, enabling ultrafast real-time spectroscopic measurements. Following our earlier work on the theory of ADFT, here we study the effect of noise on ADFT. We derive the noise figure of ADFT and discuss its dependence on various parameters.

  10. Advanced Techniques for Fourier Transform Wavefront Reconstruction

    SciTech Connect

    Poyneer, L A

    2002-08-05

    The performance of Fourier transform (FT) reconstructors in large adaptive optics systems with Shack-Hartmann sensors and a deformable mirror is analyzed. FT methods, which are derived for point-based geometries, are adapted for use on the continuous systems. Analysis and simulation show how to compensate for effects such as misalignment of the deformable mirror and wavefront sensor gain. Further filtering methods to reduce noise and improve performance are presented. All these modifications can be implemented at the filtering stage, preserving the speed of FT reconstruction. Simulation of a large system shows how compensated FT methods can have equivalent or better performance to slower vector-matrix-multiply reconstructions.

  11. Fourier transform infrared spectroscopy of deuterated proteins

    NASA Astrophysics Data System (ADS)

    Marcano O., A.; Markushin, Y.; Melikechi, N.; Connolly, D.

    2008-08-01

    We report on Fourier transform spectra of deuterated proteins: Bovine Serum Albumin, Leptin, Insulin-like Growth Factor II, monoclonal antibody to ovarian cancer antigen CA125 and Osteopontin. The spectra exhibit changes in the relative amplitude and spectral width of certain peaks. New peaks not present in the non-deuterated sample are also observed. Ways for improving the deuteration of proteins by varying the temperature and dilution time are discussed. We propose the use of deuterated proteins to increase the sensitivity of immunoassays aimed for early diagnostic of diseases most notably cancer.

  12. Time-of-flight Fourier UCN spectrometer

    NASA Astrophysics Data System (ADS)

    Kulin, G. V.; Frank, A. I.; Goryunov, S. V.; Kustov, D. V.; Geltenbort, P.; Jentschel, M.; Lauss, B.; Schmidt-Wellenburg, P.

    2016-05-01

    We describe a new time-of-flight Fourier spectrometer for investigation of UCN diffraction by a moving grating. The device operates in the regime of a discrete set of modulation frequencies. The results of the first experiments show that the spectrometer may be used for obtaining UCN energy spectra in the energy range of 60 - 200 neV with a resolution of about 5 neV. The accuracy of determination of the line position was estimated to be several units of 10-10 eV.

  13. Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

    PubMed

    Cunin, R; Rani, C S; Van Vliet, F; Wild, J R; Wales, M

    1999-12-17

    The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified. PMID:10600394

  14. Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.

    PubMed

    Revell, D F; Cummings, N J; Baker, K C; Collins, M E; Taylor, M A; Sumner, I G; Pickersgill, R W; Connerton, I F; Goodenough, P W

    1993-05-30

    We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega. PMID:7684720

  15. Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex

    PubMed Central

    Gomes, Marco Túlio Ribeiro; Teixeira, Raphael Dias; Ribeiro, Henrique de Assis Lopes; Turchetti, Andréia Pereira; Junqueira, Caroline Furtado; Lopes, Míriam Tereza Paz; Salas, Carlos Edmundo; Nagem, Ronaldo Alves Pinto

    2008-01-01

    Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P21), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 Å, β = 96.81°, and diffract X-rays to 1.8 Å resolution. PMID:18540057

  16. Inhibition of antigen- and lectin-induced proliferation of rat spleen cells by a Taenia taeniaeformis proteinase inhibitor.

    PubMed Central

    Leid, R W; Suquet, C M; Perryman, L E

    1984-01-01

    Rat splenic lymphocytes, cultured in vitro for 3 days in the presence of a larval cestode proteinase inhibitor, exhibited a marked suppression of proliferation when stimulated with Con A, PHA, PWM and ovalbumin. Reduced responsiveness was observed over a full range of concentrations of Con A (16-fold), PHA (50-fold), PWM (four-fold) and ovalbumin (16-fold). These results indicated that the inhibitory action could not be overcome by increasing the mitogen or antigen doses beyond optimal levels. This suppressive effect disappeared when the Taenia taeniaeformis proteinase inhibitor was added 20 h after the initiation of culture, suggesting that the inhibitor affects lymphocyte blastogenesis during the early stages of lymphocyte activation. PMID:6744668

  17. Optical interpretation of a complex-order Fourier transform.

    PubMed

    Shih, C C

    1995-05-15

    It is demonstrated that the definition of a fractional-order Fourier transform can be extended into the complexorder regime. A complex-order Fourier transform deals with the imaginary part as well as the real part of the exponential function in the integral. As a result, while the optical implementation of fractional-order Fourier transform involves gradient-index media or spherical lenses, the optical interpretation of complex-order Fourier transform is practically based on the convolution operation and Gaussian apertures. The beam width of a Gaussian beam subjected to the complex-order Fourier transform is obtained analytically with the ABCD matrix approach. PMID:19859464

  18. Alternating multivariate trigonometric functions and corresponding Fourier transforms

    NASA Astrophysics Data System (ADS)

    Klimyk, A. U.; Patera, J.

    2008-04-01

    We define and study multivariate sine and cosine functions, symmetric with respect to the alternating group An, which is a subgroup of the permutation (symmetric) group Sn. These functions are eigenfunctions of the Laplace operator. They determine Fourier-type transforms. There exist three types of such transforms: expansions into corresponding sine-Fourier and cosine-Fourier series, integral sine-Fourier and cosine-Fourier transforms, and multivariate finite sine and cosine transforms. In all these transforms, alternating multivariate sine and cosine functions are used as a kernel.

  19. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

    PubMed Central

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure. PMID:25136511

  20. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    PubMed Central

    Björklund, H V; Johansson, T R; Rinne, A

    1997-01-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor. PMID:9188644