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Sample records for modulates acidic proteases

  1. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    , directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection......Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

  2. Improvement of acid protease production by a mixed culture of ...

    African Journals Online (AJOL)

    The synthesis of acid protease by Aspergillus oryzae AS3042 was enhanced significantly with the mixed culture of Aspergillus niger SL-09 using solid-state fermentation technique. The influence of carbon sources, nitrogen sources and the addition of phytic acid on acid protease production were investigated. The enzyme ...

  3. Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

    Science.gov (United States)

    Kernaghan, Gavin; Mayerhofer, Michael

    2017-01-01

    This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

  4. Some physicochemical properties of acid protease produced during ...

    African Journals Online (AJOL)

    The growth of Aspergillus niger (NRRL 1785) was investigated and monitored over a five-day fermentation period. Acid protease synthesis by this fungus was also investigated during the period. The effect of growth of Aspergillus niger on acid protease synthesis was determined. Some of the physicochemical properties of ...

  5. Extracellular acid protease from Aspergillus niger I1: purification and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... A new strain of Aspergillus niger producing acid protease was isolated and identified by universal primers NL1 and .... Media were autoclaved at 120°C for 20 min. ... molecular weight calibration kit as markers consisting of bovine ... then removed by washing the gel three times with 100 mM ..... New York.

  6. A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

    Science.gov (United States)

    Riestra, Angelica M.; Gandhi, Shiv; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J.

    2015-01-01

    Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis. PMID:26684303

  7. Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

    Directory of Open Access Journals (Sweden)

    Amit K. Sharma

    2016-12-01

    Full Text Available A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

  8. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

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    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  9. Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-acyl aromatic alpha-amino acids.

    Science.gov (United States)

    Stranix, Brent R; Lavallée, Jean-François; Sévigny, Guy; Yelle, Jocelyn; Perron, Valérie; LeBerre, Nicholas; Herbart, Dominik; Wu, Jinzi J

    2006-07-01

    A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

  10. Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

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    Matthew J Kesic

    Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

  11. Physical and chemical properties of the acid protease from ...

    African Journals Online (AJOL)

    samsung

    2016-03-02

    Mar 2, 2016 ... is the principal set of biochemical changes during ... coagulating ability by analysis of the products of casein ... for protease activity, milk-clotting activity and protein content. ..... Figure 5, the content of casein components decreased in .... Purification, caracterization, molecular cloning and modelling of its.

  12. Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules.

    Science.gov (United States)

    An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin

    2015-10-01

    Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

  13. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1975-12-01

    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  14. The Effect of Exogenous Protease in Broiler Diets on the Apparent Ileal Digestibility of Amino Acids and on Protease Activity in Jejunum

    Directory of Open Access Journals (Sweden)

    Vojtěch Rada

    2016-01-01

    Full Text Available The objective of this study was to evaluate the effect of a mono-component commercial serine protease supplement in broiler diets on apparent ileal amino acid digestibility and protease activity. A total of 150 male (28 d old ROSS 308 were randomly placed into 30 battery pens and divided into 5 treatment groups with 6 replicates each. The experiment was performed for 7 days. Five dietary treatments were used: 2 standard protein diets without (SP and with protease (SP + P formulated 20.7 % CP, 2 lower-protein diets (19.9 % CP without (LP and with protease (LP + P and one lower‑protein diet with protease and with doubled rapeseed meal (RSM content (SP-RSM + P compared with the other treatments. Lower-protein diets were formulated with a 4 % decrease in the relative CP value compared with the standard protein diet. Enzyme protease was added to the diets at a concentration of 200 ppm (15,000 PROT units per kg. The diets contained 0.3 % Cr2O3 to facilitate the estimation of apparent AA digestibility and overall apparent ileal crude protein digestibility. Mono-component protease had no effect on apparent ileal AA digestibility or jejunum protease activity if diets contained the same level of RSM. The supplement of exogenous protease did not affect (P > 0.05 the apparent ileal AA digestibility coefficients if a higher RSM level was used. The CP level influenced (P < 0.05 only the coefficients of the apparent ileal AA digestibility of Pro and Arg. The RSM level (P < 0.01 had significant effects on protease activity in the jejunum.

  15. Production of rennin-like acid protease by Mucor pusillus through submerged fermentation

    International Nuclear Information System (INIS)

    Daudi, S.; Mukhtar, H.; Rehman, A.U.; Haq, I.U.

    2015-01-01

    The present study is concerned with the isolation and screening of Mucor species for the production of acid protease in shake flasks. Out of eight mould cultures evaluated, five were isolated from soil and three were provided from the Institute of Industrial Biotechnology, Government College University, Lahore. Of all the isolates tested, Mucor pusillus IHS6 was found to be the best producer of rennin-like acid protease producing 75 U/ml of the enzyme. Different agricultural byproducts were evaluated as fermentation substrates and maximum enzyme synthesis (61 U/ml) was obtained when rapeseed meal was used as a substrate. Optimum pH and fermentation period for the production of protease were 5.5 (56U/ml) and 72 hrs (55U/ml), respectively. The production of protease by Mucor pusillus IHS6 was also studied by adding different carbon and nitrogen sources to the fermentation medium. Fructose at a concentration of 1.5% (66 U/ml) and yeast extract at a concentration of 2% (68.2 U/ml) and ammonium chloride at a concentration of 0.1% (67U/ml) were found to be the best carbon and nitrogen (organic and inorganic) sources respectively. Spore inoculum at a concentration of 1% (68.4 U/ml) was found to be the best for protease production by Mucor pusillus. The fermentation broth was found to have strong milk clotting activity with 200 RU. (author)

  16. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

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    Honek John F

    2005-10-01

    Full Text Available Abstract Background The alkaline protease from Pseudomonas aeruginosa (AprA is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM, into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.

  17. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    Science.gov (United States)

    Walasek, Paula; Honek, John F

    2005-01-01

    Background The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease. PMID:16221305

  18. Inhibition of proteases activity in intestine needs a sustainable acidic environment rather than a transient.

    Science.gov (United States)

    Xing, Chang; Xing, Jin-Feng; Ge, Zhi-Qiang

    2017-10-01

    α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.

  19. A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

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    Siniša Urban

    2014-09-01

    Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

  20. A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

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    Abirami Kugadas

    2016-08-01

    Full Text Available AbstractThe ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP, the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophage and MAC-T cells and coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increase bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5 conditions. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

  1. Kinetics of leather dyeing pretreated with enzymes: role of acid protease.

    Science.gov (United States)

    Kanth, Swarna Vinodh; Venba, Rajangam; Jayakumar, Gladstone Christopher; Chandrababu, Narasimhan Kannan

    2009-04-01

    In the present investigation, kinetics of dyeing involving pretreatment with acid protease has been presented. Application of acid protease in dyeing process resulted in increased absorption and diffusion of dye into the leather matrix. Enzyme treatment at 1% concentration, 60 min duration and 50 degrees C resulted in maximum of 98% dye exhaustion and increased absorption rate constants. The final exhaustion (C(infinity)) for the best fit of CI Acid Black 194 dye has been 98.5% with K and r2 values from the modified Cegarra-Puente isotherm as 0.1033 and 0.0631. CI Acid Black 194 being a 2:1 metal complex acid dye exhibited higher absorption rate than the acid dye CI Acid Black 210. A reduction in 50% activation energy calculated from Arrhenius equation has been observed in enzyme assisted dyeing process of both the dyes that substantiates enhanced dye absorption. The absorption rate constant calculated with modified Cegarra-Puente equation confirm higher rate constants and faster kinetics for enzyme assisted dyeing process. Enzyme treated leather exhibited richness of color and shade when compared with control. The present study substantiates the essential role of enzyme pretreatment as an eco-friendly leather dyeing process.

  2. Advances in protease engineering for laundry detergents.

    Science.gov (United States)

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    Science.gov (United States)

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  4. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring

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    Mohamed Amine Gomri

    2018-04-01

    Full Text Available Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  5. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring.

    Science.gov (United States)

    Gomri, Mohamed Amine; Rico-Díaz, Agustín; Escuder-Rodríguez, Juan-José; El Moulouk Khaldi, Tedj; González-Siso, María-Isabel; Kharroub, Karima

    2018-04-12

    Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  6. Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus.

    Science.gov (United States)

    Souza, Paula Monteiro; Aliakbarian, Bahar; Filho, Edivaldo Ximenes Ferreira; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa; Converti, Attilio; Perego, Patrizia

    2015-11-01

    The kinetics of a thermostable extracellular acid protease produced by an Aspergillus foetidus strain was investigated at different pH, temperatures and substrate concentrations. The enzyme exhibited maximal activity at pH 5.0 and 55°C, and its irreversible deactivation was well described by first-order kinetics. When temperature was raised from 55 to 70°C, the deactivation rate constant increased from 0.018 to 5.06h(-1), while the half-life decreased from 37.6 to 0.13h. The results of activity collected at different temperatures were then used to estimate, the activation energy of the hydrolysis reaction (E*=19.03kJ/mol) and the standard enthalpy variation of reversible enzyme unfolding (ΔH°U=19.03kJ/mol). The results of residual activity tests carried out in the temperature range 55-70°C allowed estimating the activation energy (E(*)d=314.12kJ/mol), enthalpy (311.27≤(ΔH°d≤311.39kJ/mol), entropy (599.59≤ΔS(*)d≤610.49kJ/mol K) and Gibbs free energy (103.18≤ΔG(*)d≤113.87kJ/mol) of the enzyme irreversible denaturation. These thermodynamic parameters suggest that this new protease is highly thermostable and could be important for industrial applications. To the best of our knowledge, this is the first report on thermodynamic parameters of an acid protease produced by A. foetidus. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

    Czech Academy of Sciences Publication Activity Database

    Grantz Šašková, Klára; Kožíšek, Milan; Stray, K.; Jong de, D.; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; Nijhuis, M.; Cihlář, T.; Konvalinka, Jan

    2014-01-01

    Roč. 88, č. 6 (2014), s. 3586-3590 ISSN 0022-538X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : virus type-1 protease * antiviral activity * drug resistance Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

  8. Modulating effects of the protease inhibitor Antipain on x-ray induced transformations

    International Nuclear Information System (INIS)

    Borek, C.; Miller, R.C.

    1979-01-01

    Protease inhibitors have been shown to inhibit the expression of mutations in bacteria and to inhibit the tumor-promoting effect of phorbol esters in mice. We have investigated the effect of the protease inhibitor Antipain on cell transformation by x-irradiation in two in vitro systems; namely short-term cultures of freshly explanted hamster embryo cells and in the 10T1/2 cell line derived and cloned from C3H mouse embryo

  9. Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

    Science.gov (United States)

    Goel, C; Kalra, N; Dwarakanath, B S; Gaur, S N; Arora, N

    2015-05-01

    Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses. © 2014 British Society for Immunology.

  10. Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Aditi Narendra Borkar

    Full Text Available Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

  11. Isolation of Lactic Acid Bacteria That Produce Protease and Bacteriocin-Like Substance From Mud Crab (Scylla sp. Digestive Tract (Isolasi Bakteri Asam Laktat yang Menghasilkan Protease dan Senyawa Bacteriocin-Like dari Saluran Pencernaan Kepiting

    Directory of Open Access Journals (Sweden)

    Heru Pramono

    2015-03-01

    Kata kunci: Bakteri Asam Laktat, Bakteriosin-like substance, Protease, Scylla  sp. Digestive tract is complex environment consist of large amount of bacteria’s species. Fish intestine bacteria consist of aerobic or facultative anaerob bacteria which can produce antibacterial and enzym. The objectives of this research were to isolated lactic acid bacteria that produce bacteriocin-like and protease from mud crab digestive tract. Isolation and characterization of isolates were conducted employing media MRS.  Neutralized cell free supernatant of isolates were tested using disc diffusion agar of against pathogenic and spoilage bacteria to indicate bacteriocin-like-producing lactic acid bacteria. Protease-producing isolate was tested using disc diffusion method in casein agar. Among a hundred isolates, 96 isolates were showed clear zone in MRS+CaCO3,, catalase negative, and Gram positive bacteria. Thirty four isolates produced protease and only four isolates (i.e. IKP29, IKP30, IKP52, and IKP94 showed strong inhibition against pathogenic and spoilage bacteria. There were three patterns of inhibition among three isolates against Bacillus subtilis, Staphylococcus aureus, Eschericia coli, and Salmonella sp. All three isolates showed potential uses for produce starter culture for fishery product fermentation purpose. This is the first report of isolation lactic acid bacteria that produced protease and bacteriocin-like from digestive tract of mud crab. Keywords: Lactic acid bacteria, Bacteriocin-like substance, Protease, Scylla  sp.

  12. Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1976-01-01

    The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  13. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7.

    Science.gov (United States)

    Lario, Luciana Daniela; Chaud, Luciana; Almeida, María das Graças; Converti, Attilio; Durães Sette, Lara; Pessoa, Adalberto

    2015-11-01

    The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  14. Selection of protease for increased solubilization of protein-derived thiols during mashing with limited release of free amino acids in beer

    DEFF Research Database (Denmark)

    Murmann, Anne Nordmark; Lunde, Christina; Lund, Marianne Nissen

    2016-01-01

    Extraction of protein-derived thiols by protease treatment during mashing for improvement of flavor stability in beer has previously been shown to cause concomitant increase in free amino acid concentrations and thereby increased levels of unwanted Maillard reaction products during aging. The pre......Extraction of protein-derived thiols by protease treatment during mashing for improvement of flavor stability in beer has previously been shown to cause concomitant increase in free amino acid concentrations and thereby increased levels of unwanted Maillard reaction products during aging...... of a protease with a higher temperature optimum dosed at only 3 mg of enzyme/kg of malt, it is possible to increase thiol concentrations in wort by 30% and with only a maximum 10% increase in amino acid concentration compared with a control. Pilot brewing showed that beer brewed with addition of protease...... stability during storage could not be evaluated. Overall, similar brewing and sensory characteristics were obtained compared with a control beer brewed without addition of protease. Foam stability was decreased by protease treatment, and formation of haze was reduced by protease treatment....

  15. A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

    Science.gov (United States)

    Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Cruz de Carvalho, Maria H

    2013-11-15

    A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits. Copyright © 2013 Elsevier GmbH. All rights reserved.

  16. Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

    NARCIS (Netherlands)

    Kaman, W.E.; Voskamp-Visser, I.; de Jongh, D.M.C.; Endtz, H.P.; van Belkum, A.; Hays, J.P.; Bikker, F.J.

    2013-01-01

    Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these

  17. Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

    Science.gov (United States)

    Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

    2015-07-01

    Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. [Roles of protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, in modulation of exocrine gland functions].

    Science.gov (United States)

    Nishikawa, Hiroyuki

    2006-07-01

    Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal extracellular tethered ligand that presumably binds to the extracellular loop 2 of the receptor itself. PAR-2 is widely distributed in the mammalian body and plays various roles in biological events in the cardiovascular, respiratory, alimentary, and central neurons systems. PAR-2-activating peptides administered systemically to mice and rats trigger prompt salivation in vivo. In an in vitro study, PAR-2 agonists including the endogenous PAR-2 activator trypsin induce secretion of amylase and mucin from isolated rat parotid glands and sublingual glands, respectively. PAR-2-activating peptides administered systemically also modulate pancreatic exocrine secretion in vivo as well as in vitro. In the gastric mucosa, PAR-2 stimulation enhances secretion of mucus and pepsinogen and suppresses acid secretion. Tear secretion can also be caused by PAR-2-related peptides in PAR-2-dependent and -independent manners. PAR-2 thus plays a general or key role in the regulation of exocrine secretion. This review focuses on the physiologic and/or pathophysiologic roles of PAR-2 in glandular exocrine secretion. The possibility of PAR-2 as a target for drug development is also discussed.

  19. Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

    International Nuclear Information System (INIS)

    Qasim, Mohammad A.; Song, Jikui; Markley, John L.; Laskowski, Michael

    2010-01-01

    Research highlights: → Large pK shifts in ionizable groups when buried in the protein interior. → Substrate dependent shifts in pH optimum for serine proteases. → Lys side chain is a stronger acid in serine protease S 1 pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S 1 pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

  20. Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

    Science.gov (United States)

    Rout, Manoj Kumar; Hosur, Ramakrishna V

    2009-02-01

    Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

  1. Macrophage migration inhibitory factor (MIF) modulates trophic signaling through interaction with serine protease HTRA1

    DEFF Research Database (Denmark)

    Fex Svenningsen, Åsa; Loering, Svenja; Sørensen, Anna Lahn

    2017-01-01

    Macrophage migration inhibitory factor (MIF), a small conserved protein, is abundant in the immune- and central nervous system (CNS). MIF has several receptors and binding partners that can modulate its action on a cel-lular level. It is upregulated in neurodegenerative diseases and cancer although...

  2. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    Science.gov (United States)

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

  3. Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

    Science.gov (United States)

    de Magalhães, Mariana T Q; Mambelli, Fábio S; Santos, Bruno P O; Morais, Suellen B; Oliveira, Sergio C

    2018-03-31

    Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase

    DEFF Research Database (Denmark)

    Pedersen, B K; Kharazmi, A; Theander, T G

    1987-01-01

    The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA......-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods...

  5. Effects of Coated Compound Proteases on Apparent Total Tract Digestibility of Nutrients and Apparent Ileal Digestibility of Amino Acids for Pigs.

    Science.gov (United States)

    Pan, L; Zhao, P F; Yang, Z Y; Long, S F; Wang, H L; Tian, Q Y; Xu, Y T; Xu, X; Zhang, Z H; Piao, X S

    2016-12-01

    Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: 20.14±1.71 kg) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (pdigestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (pdigestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment.

  6. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases

    DEFF Research Database (Denmark)

    Batten, MR; Senior, BW; Kilian, Mogens

    2003-01-01

    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either...... side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial...... effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement...

  7. Immune and cell modulation by amino acids.

    Science.gov (United States)

    Roth, Erich

    2007-10-01

    Sir David Cuthbertson was the first to define metabolic alterations in post-aggression syndrome (PAS). From basic measurements of nitrogen loss and total protein synthesis/degradation, the current research has moved to genomics, proteomics and metabolomics. In this respect, first evidence was accumulated for the influence of acute catabolism, immobilisation by bed rest and sarcopenia of old age on the muscle-cell genome and proteome. Moreover, in post-aggression syndrome specific amino acids such as glutamine, arginine, glycine, taurine, tryptophan and cysteine are used for cell and immune modulation. Our laboratory has focused on the regulative capacity of glutamine. Glutamine deficiency as found in post-aggression syndrome reduces lymphocyte proliferation, alters monocyte/macrophage activity, decreases the formation of heat-shock proteins, stimulates cell apoptosis, shifts the cellular redox potential by altering the glutathione synthesis and increases the activity of the AMPK system. Investigating the molecular effect of glutamine on Hsp 70 induction, we tested the glutamine dependence on the formation of transfer-RNA and of heat-shock factor 1 (HSF 1), and on transcription and translation of Hsp 70. We could demonstrate that glutamine stabilises the mRNA of Hsp 70 thereby prolonging its half-life. The lecture also discusses the principal molecular targets of administered arginine, glycine, cysteine, taurine and tryptophan.

  8. Presenilin is the molecular target of acidic γ-secretase modulators in living cells.

    Directory of Open Access Journals (Sweden)

    Thorsten Jumpertz

    Full Text Available The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs, which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC(50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN, the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.

  9. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

    DEFF Research Database (Denmark)

    Senior, BW; Batten, MR; Kilian, Mogens

    2002-01-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were const...... constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases....

  10. A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue.

    Science.gov (United States)

    Wang, S Y; Gudas, L J

    1990-09-15

    We have previously isolated several cDNA clones specific for mRNA species that increase in abundance during the retinoic acid-associated differentiation of F9 teratocarcinoma stem cells. One of these mRNAs, J6, encodes a approximately 40 kDa protein as assayed by hybrid selection and in vitro translation (Wang, S.-Y., LaRosa, G., and Gudas, L. J. (1985) Dev. Biol. 107, 75-86). The time course of J6 mRNA expression is similar to those of both laminin B1 and collagen IV (alpha 1) messages following retinoic acid addition. To address the functional role of this protein, we have isolated a full-length cDNA clone complementary to this approximately 40-kDa protein mRNA. Sequence analysis reveals an open reading frame of 406 amino acids (Mr 45,652). The carboxyl-terminal portion of this predicted protein contains a region that is homologous to the reactive sites found among members of the serpin (serine protease inhibitor) family. The predicted reactive site (P1-P1') of this J6 protein is Arg-Ser, which is the same as that of antithrombin III. Like ovalbumin and human monocyte-derived plasminogen activator inhibitor (mPAI-2), which are members of the serpin gene family, the J6 protein appears to have no typical amino-terminal signal sequence.

  11. Effect of mefenamic acid on the immunity and hemostatic system of cancer patients and on the activity of cathepsin D-like protease in colonic cancer tissue

    International Nuclear Information System (INIS)

    Klyachkin, B.M.; Basargin, S.T.; Timofeev, I.V.; Khaliulin, Yu.G.; Dorofeev, S.A.; Alekseenko, L.D.; Gumenyuk, M.L.

    1992-01-01

    The study of the effect of sodium mefenaminate on radiation resistance of mice yielded positive results. Clinical investigations showed mefenamic acid to decrease the activity of cathepsin D-like protease in colonic cancer tissue. The acid field to affect the proteolytic activity of the normal mucosa. It revealed an immunomodulating activity and influenced the hemostatic system which usually manifested itself in amelioration of hypercoagulation

  12. Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

    NARCIS (Netherlands)

    Bossers, A.; Belt, P.B.G.M.; Raymond, G.J.; Caughey, B.; Vries, de R.; Smits, M.

    1997-01-01

    Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system.

  13. Nitrated Fatty Acids Reverse Cigarette Smoke-Induced Alveolar Macrophage Activation and Inhibit Protease Activity via Electrophilic S-Alkylation.

    Science.gov (United States)

    Reddy, Aravind T; Lakshmi, Sowmya P; Muchumarri, Ramamohan R; Reddy, Raju C

    2016-01-01

    Nitrated fatty acids (NFAs), endogenous products of nonenzymatic reactions of NO-derived reactive nitrogen species with unsaturated fatty acids, exhibit substantial anti-inflammatory activities. They are both reversible electrophiles and peroxisome proliferator-activated receptor γ (PPARγ) agonists, but the physiological implications of their electrophilic activity are poorly understood. We tested their effects on inflammatory and emphysema-related biomarkers in alveolar macrophages (AMs) of smoke-exposed mice. NFA (10-nitro-oleic acid or 12-nitrolinoleic acid) treatment downregulated expression and activity of the inflammatory transcription factor NF-κB while upregulating those of PPARγ. It also downregulated production of inflammatory cytokines and chemokines and of the protease cathepsin S (Cat S), a key mediator of emphysematous septal destruction. Cat S downregulation was accompanied by decreased AM elastolytic activity, a major mechanism of septal destruction. NFAs downregulated both Cat S expression and activity in AMs of wild-type mice, but only inhibited its activity in AMs of PPARγ knockout mice, pointing to a PPARγ-independent mechanism of enzyme inhibition. We hypothesized that this mechanism was electrophilic S-alkylation of target Cat S cysteines, and found that NFAs bind directly to Cat S following treatment of intact AMs and, as suggested by in silico modeling and calculation of relevant parameters, elicit S-alkylation of Cys25 when incubated with purified Cat S. These results demonstrate that NFAs' electrophilic activity, in addition to their role as PPARγ agonists, underlies their protective effects in chronic obstructive pulmonary disease (COPD) and support their therapeutic potential in this disease.

  14. Synergistic action of protease-modulating matrix and autologous growth factors in healing of diabetic foot ulcers. A prospective randomized trial.

    Science.gov (United States)

    Kakagia, Despoina D; Kazakos, Konstantinos J; Xarchas, Konstantinos C; Karanikas, Michael; Georgiadis, George S; Tripsiannis, Gregory; Manolas, Constantinos

    2007-01-01

    This study tests the hypothesis that addition of a protease-modulating matrix enhances the efficacy of autologous growth factors in diabetic ulcers. Fifty-one patients with chronic diabetic foot ulcers were managed as outpatients at the Democritus University Hospital of Alexandroupolis and followed up for 8 weeks. All target ulcers were > or = 2.5 cm in any one dimension and had been previously treated only with moist gauze. Patients were randomly allocated in three groups of 17 patients each: Group A was treated only with the oxidized regenerated cellulose/collagen biomaterial (Promogran, Johnson & Johnson, New Brunswick, NJ), Group B was treated only with autologous growth factors delivered by Gravitational Platelet Separation System (GPS, Biomet), and Group C was managed by a combination of both. All ulcers were digitally photographed at initiation of the study and then at change of dressings once weekly. Computerized planimetry (Texas Health Science Center ImageTool, Version 3.0) was used to assess ulcer dimensions that were analyzed for homogeneity and significance using the Statistical Package for Social Sciences, Version 13.0. Post hoc analysis revealed that there was significantly greater reduction of all three dimensions of the ulcers in Group C compared to Groups A and B (all P<.001). Although reduction of ulcer dimensions was greater in Group A than in Group B, these differences did not reach statistical significance. It is concluded that protease-modulating dressings act synergistically with autologous growth factors and enhance their efficacy in diabetic foot ulcers.

  15. Effect of hydrolysis enzymatic process of corn using protease crude (Rhizopus oligosporus-C1) to produce corn hydrolisate rich folic acid

    Science.gov (United States)

    Maryati, Yati; Susilowati, Agustine; Melanie, Hakiki; Lotulung, Puspa D.

    2017-11-01

    Corn hydrolyzate (Zea mays L) as a functional food fortificant derived from natural folic acid has been evaluated through a hydrolysis process using protease enzyme Rhizopus oligosporus strain C1. Enzymatic hydrolysis was carried out on two types of corn; yellow and white pearl variety corn, at concentration of protease enzyme (rough) 0.025; 0.125; and 0.225% (v/w of soluble nixtamal corn protein) with a hydrolysis time of 24 h at 30 °C, and pH 5.0. The results showed that the concentration of protease enzymes can increase the folic acid to the optimum condition, from the beginning to the end of the process time. Folic acid optimization of hydrolysis results in each corn was at the concentration of protease enzyme 0.225% (v/w of soluble nixtamal corn protein) in white corn and yellow corn at 24 hours hydrolysis, with folic acid composition, 283.56 µg/mL and 412.52 µg/mL, 1.07 and 1.04 mg/mL of soluble proteins, proteolytic activity 2.09 and 2.06 U/mL, total solids of 21.74 and 17.85%, total sugars of 0.56 and 2.22 mg/mL, and reducing sugar 91.72 and 48.47 mg/mL. In this condition, the increase of optimum folic acid for white corn was 33.57% and for yellow corn was 71.60% after hydrolysis.

  16. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    Czech Academy of Sciences Publication Activity Database

    Maarseveen van, N. M.; Andersson, Dan; Lepšík, Martin; Fun, A.; Schipper, P. J.; Jong de, D.; Boucher, Ch. A. B.; Nijhuis, M.

    2012-01-01

    Roč. 9, č. 29 (2012), s. 1-7 ISSN 1742-4690 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Grant - others:Dutch AIDS Fund(XE) 2006028; (NWO) VIDI(XE) 91796349 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 * protease * Gag * resistance * cleavage Subject RIV: CE - Biochemistry Impact factor: 5.657, year: 2012

  17. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

    Directory of Open Access Journals (Sweden)

    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  18. Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor, Lopinavir

    Directory of Open Access Journals (Sweden)

    Mitesh Patel

    2014-04-01

    Full Text Available Poor systemic concentrations of lopinavir (LPV following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp and multidrug resistance-associated proteins (MRPs and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1 and MRP2 (MDCK-MRP2 transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2.

  19. Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

    Science.gov (United States)

    Korosh, Travis; Jordan, Kelsey D; Wu, Ja-Shin; Yarlett, Nigel; Upmacis, Rita K

    2016-01-01

    Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  20. Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

    International Nuclear Information System (INIS)

    Yoon, J-Y; Szwajcer, D; Ishdorj, G; Benjaminson, P; Xiao, W; Kumar, R; Johnston, J B; Gibson, S B

    2013-01-01

    Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins

  1. Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

    Science.gov (United States)

    Hauptmann, Peter; Lehle, Ludwig

    2008-07-04

    N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

  2. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  3. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan

    2010-01-01

    Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  4. Earthworm Protease

    International Nuclear Information System (INIS)

    Pan, R.; Zhang, Z.; He, R.

    2010-01-01

    The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  5. Liberation of Desmosine and Isodesmosine as Amino Acids from Insoluble Elastin by Elastolytic Proteases

    Science.gov (United States)

    Umeda, Hideyuki; Aikawa, Masanori; Libby, Peter

    2011-01-01

    The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation. PMID:21726534

  6. The effect of exogenous protease in broiler diets on the apparent ileal digestibility of amino acids and on protease activity in jejunum

    Czech Academy of Sciences Publication Activity Database

    Rada, V.; Lichovníková, M.; Foltýn, M.; Šafařík, Ivo

    2016-01-01

    Roč. 64, č. 5 (2016), s. 1645-1652 ISSN 1211-8516 Institutional support: RVO:60077344 Keywords : chicken * crude protein * methionine * glutamic acid * rapeseed meal Subject RIV: GH - Livestock Nutrition

  7. Comparative Amino Acids Studies on Phac Synthases and Proteases as Well as Establishing a New Trend in Experimental Design

    Directory of Open Access Journals (Sweden)

    Amro Abd al fattah Amara

    2012-04-01

    Full Text Available ABSTRACT: A question addressed in this study is: why similar enzymes are classified into different subclasses? As an example, PhaC synthases are classified according to four different classes (I, II, III and IV. To answer this question we proposed that besides the catalytic residues, the overall amino acids (AAs present are responsible for the differences observed. The AAs’ composition affects the structure/function/substrate specificity (SFS of these enzymes. The differences between the classes in various PhaC synthases and proteases were analysed to support our argument. Homology and phylogenic tree of some selected PhaC synthases of different strains (representing the four classes were demonstrated. The properties of a specific class of enzyme could not be changed into those of another by changing the catalytic residues. Moreover, these differences could not be detected from the proteins’ 3D structures, despite clear differences at the AAs level. Another question was also addressed: could we benefit from the various existing protein databases in the field of biotechnology? To answer this, we introduced a model for an Experimental Design based on the information in the protein database (for strains available in our lab regarding their ability to degrade castor oil. Two enzymes in the phenol degradation pathway, phenol 2-monooxygenase and catechol 1,2-dioxygenase, and a lipase enzyme were analysed. These enzymes were screened and analysed according to the BLAST-protein database and BRENDA. The comprehensive enzyme information system compared six strains against each other, including: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis, and Geobacillus stearothermophilus. Only P. aeruginosa proved to have the three required enzymes and was suitable for the production of lipases from castor oil (crude castor oil is usually contaminated with phenol as indicated by the databases. In

  8. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    -terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

  9. Specific fatty acids as metabolic modulators in the dairy cow

    Directory of Open Access Journals (Sweden)

    J.A.A. Pires

    2008-07-01

    Full Text Available This review summarizes recent developments on the utilization of specific fatty acids to modulate bovine energy metabolism, with emphasis on the periparturient dairy cow. A number of experiments have assessed the effects of polyunsaturated fatty acids on bovine hepatic energy metabolism using in vitro and in vivo models. Treatment of hepatocytes with specific fatty acids altered energy metabolism in vitro. For example, linolenic acid seemed to decrease hepatocyte triacylglycerol accumulation. This effect was confirmed in vivo, using parenteral infusions of emulsions derived from different fat sources to feed-restricted non-lactating cows. Additionally, polyunsaturated fatty acids can increase whole body response to insulin, potentially enhancing antilipolytic effects of insulin and muscle protein anabolism in the bovine. There is limited literature on the effects of feeding fat sources rich in omega-3 polyunsaturated fatty acids, such as fish oil and linseed oil, on metabolism of periparturient dairy cows. Available research has yielded conflicting results which need further clarification. On the other hand, specific isomers of conjugated linoleic acid consistently induce milk fat depression and are able to decrease energy export in milk by periparturient dairy cows. Nonetheless, research is still needed to assess whether these effects will ultimately benefit productivity and health status of periparturient dairy cows. Limitations of available methods to protect fatty acids from ruminal biohydrogenation are also addressed.

  10. Retinoic Acid and Its Role in Modulating Intestinal Innate Immunity

    Directory of Open Access Journals (Sweden)

    Paulo Czarnewski

    2017-01-01

    Full Text Available Vitamin A (VA is amongst the most well characterized food-derived nutrients with diverse immune modulatory roles. Deficiency in dietary VA has not only been associated with immune dysfunctions in the gut, but also with several systemic immune disorders. In particular, VA metabolite all-trans retinoic acid (atRA has been shown to be crucial in inducing gut tropism in lymphocytes and modulating T helper differentiation. In addition to the widely recognized role in adaptive immunity, increasing evidence identifies atRA as an important modulator of innate immune cells, such as tolerogenic dendritic cells (DCs and innate lymphoid cells (ILCs. Here, we focus on the role of retinoic acid in differentiation, trafficking and the functions of innate immune cells in health and inflammation associated disorders. Lastly, we discuss the potential involvement of atRA during the plausible crosstalk between DCs and ILCs.

  11. Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

    Science.gov (United States)

    Martinez-Moreno, Julio M; Herencia, Carmen; Montes de Oca, Addy; Muñoz-Castañeda, Juan R; Rodríguez-Ortiz, M Encarnación; Díaz-Tocados, Juan M; Peralbo-Santaella, Esther; Camargo, Antonio; Canalejo, Antonio; Rodriguez, Mariano; Velasco-Gimena, Francisco; Almaden, Yolanda

    2016-03-01

    Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs. © FASEB.

  12. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  13. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao

    2018-02-16

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named \\'tamarillin\\'.

  14. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao; Scott, Ken; Hemar, Yacine; Zhang, Huoming; Otter, Don

    2018-01-01

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

  15. A mild pulsed electric field condition that improves acid tolerance, growth, and protease activity of Lactobacillus acidophilus LA-K and Lactobacillus delbrueckii subspecies bulgaricus LB-12.

    Science.gov (United States)

    Najim, N; Aryana, Kayanush J

    2013-06-01

    Pulsed electric field (PEF) processing involves the application of pulses of voltage for less than 1 s to fluid products placed between 2 electrodes. The effect of mild PEF on beneficial characteristics of probiotic bacteria Lactobacillus acidophilus and Lactobacillus delbrueckii ssp. bulgaricus is not clearly understood. The objective of this study was to determine the influence of mild PEF conditions on acid tolerance, growth, and protease activity of Lb. acidophilus LA-K and Lactobacillus delbrueckii ssp. bulgaricus LB-12. A pilot plant PEF system (OSU-4M; The Ohio State University, Columbus) was used. The PEF treatments were positive square unipolar pulse width of 3 µs, pulse period of 0.5s, electric field strength of 1 kV/cm, delay time of 20 µs, flow rate of 60 mL/min, and 40.5°C PEF treatment temperature. Both Lb. acidophilus LA-K and Lb. bulgaricus LB-12 subjected to mild PEF conditions were acid tolerant until the end of the 120 min of incubation, unlike the Lb. bulgaricus control, which was not acid tolerant after 30 min. The mild PEF-treated Lb. acidophilus LA-K and Lb. bulgaricus LB-12 reached the logarithmic phase of growth an hour earlier than the control. Mild PEF conditions studied significantly improved acid tolerance, exponential growth, and protease activity of both Lb. acidophilus LA-K and Lb. bulgaricus LB-12 compared with the control. The mild PEF conditions studied can be recommended for pretreating cultures to enhance these desirable attributes. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Mosaic serine proteases in the mammalian central nervous system.

    Science.gov (United States)

    Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

    2008-01-01

    We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

  17. Co-evolution of insect proteases and plant protease inhibitors.

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

    Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

  18. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    user

    2013-03-20

    Mar 20, 2013 ... Full Length Research Paper. Purification and ... ting into small peptides and free amino acids, which can ... Isolated strain was cultured in synthetic medium- casein (SMC; ... Protease activity was assayed by sigma's non-specific protease ... following buffers: 0.05 M citrate-phosphate buffer (pH 5 to 6), Tris-.

  19. The cholesterol, fatty acid and triglyceride synthesis pathways regulated by site 1 protease (S1P) are required for efficient replication of severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Urata, Shuzo; Uno, Yukiko; Kurosaki, Yohei; Yasuda, Jiro

    2018-06-12

    Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection. Copyright © 2018. Published by Elsevier Inc.

  20. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease.

    Directory of Open Access Journals (Sweden)

    Gangwei Ou

    Full Text Available BACKGROUND: Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s to induce interleukin-8 (IL-8 production by human intestinal epithelial cells. The aim was to identify the secreted component(s of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

  1. High throughput in vivo protease inhibitor selection platform

    DEFF Research Database (Denmark)

    2017-01-01

    The invention relates to a recombinant microbial cell comprising a selection platform for screening for a protease inhibitor, wherein the platform comprises transgenes encoding a protease having selective peptide bond cleavage activity at a recognition site amino acid sequence; and transgenes...... platform for screening for a protease inhibitor....

  2. Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

    Science.gov (United States)

    Kihara, A; Akiyama, Y; Ito, K

    1997-05-27

    The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

  3. Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects

    DEFF Research Database (Denmark)

    Rasmussen, Michael Højby; Juul, Anders; Kjems, Lise Lund

    2006-01-01

    , no published data exist on free IGF-I levels, acid labile subunit (ALS), or IGFBP protease activity in relation to GH release during a hypocaloric diet. The main purpose of this study was to determine free IGF-I, ALS, IGFBPs-1-4, and IGFBPs-1-3 protease activity in relation to 24-h GH release before and after......Decreased levels of GH and total IGF-I have been reported in obesity. It has been hypothesized that increased free (biologically active) IGF-I levels generated from IGF-binding protein (IGFBP) protease activity could be the mechanism for the low GH release in dieting obese subjects. However...... a short-term very low-calorie diet (VLCD)....

  4. Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects

    DEFF Research Database (Denmark)

    Rasmussen, Michael Højby; Juul, Anders; Kjems, Lise Lund

    2006-01-01

    Decreased levels of GH and total IGF-I have been reported in obesity. It has been hypothesized that increased free (biologically active) IGF-I levels generated from IGF-binding protein (IGFBP) protease activity could be the mechanism for the low GH release in dieting obese subjects. However......, no published data exist on free IGF-I levels, acid labile subunit (ALS), or IGFBP protease activity in relation to GH release during a hypocaloric diet. The main purpose of this study was to determine free IGF-I, ALS, IGFBPs-1-4, and IGFBPs-1-3 protease activity in relation to 24-h GH release before and after...... a short-term very low-calorie diet (VLCD)....

  5. Retinoic acid modulates chondrogenesis in the developing mouse cranial base.

    Science.gov (United States)

    Kwon, Hyuk-Jae; Shin, Jeong-Oh; Lee, Jong-Min; Cho, Kyoung-Won; Lee, Min-Jung; Cho, Sung-Won; Jung, Han-Sung

    2011-12-15

    The retinoic acid (RA) signaling pathway is known to play important roles during craniofacial development and skeletogenesis. However, the specific mechanism involving RA in cranial base development has not yet been clearly described. This study investigated how RA modulates endochondral bone development of the cranial base by monitoring the RA receptor RARγ, BMP4, and markers of proliferation, programmed cell death, chondrogenesis, and osteogenesis. We first examined the dynamic morphological and molecular changes in the sphenooccipital synchondrosis-forming region in the mouse embryo cranial bases at E12-E16. In vitro organ cultures employing beads soaked in RA and retinoid-signaling inhibitor citral were compared. In the RA study, the sphenooccipital synchondrosis showed reduced cartilage matrix and lower BMP4 expression while hypertrophic chondrocytes were replaced with proliferating chondrocytes. Retardation of chondrocyte hypertrophy was exhibited in citral-treated specimens, while BMP4 expression was slightly increased and programmed cell death was induced within the sphenooccipital synchondrosis. Our results demonstrate that RA modulates chondrocytes to proliferate, differentiate, or undergo programmed cell death during endochondral bone formation in the developing cranial base. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

  6. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    USER

    Keywords: Protease, lactic acid bacteria, Pediococcus acidilactici, enzyme ... confers organoleptic improvements in fermented foods ... was characterized by studying the effect of substrate ... addition of solid ammonium sulphate up to 80%.

  7. Digestive system development and study of acid and alkaline protease digestive capacities using biochemical and molecular approaches in totoaba (Totoaba macdonaldi) larvae.

    Science.gov (United States)

    Galaviz, Mario A; López, Lus M; García Gasca, Alejandra; Álvarez González, Carlos Alfonso; True, Conal D; Gisbert, Enric

    2015-10-01

    The present study aimed to describe and understand the development of the digestive system in totoaba (Totoaba macdonaldi) larvae from hatching to 40 days post-hatch (dph) from morphological and functional perspectives. At hatch, the digestive system of totoaba was undifferentiated. The anus and the mouth opened at 4 and 5 dph, respectively. During exogenous feeding, development of the esophagus, pancreas, liver and intestine was observed with a complete differentiation of all digestive organs. Expression and activity of trypsin and chymotrypsin were observed as early as at 1 dph, and increments in their expression and activity coincided with changes in food items (live and compound diets) and morpho-physiological development of the accessory digestive glands. In contrast, pepsin was detected later during development, which includes the appearance of the gastric glands between 24 and 28 dph. One peak in gene expression was detected at 16 dph, few days before the initial development of the stomach at 20 dph. A second peak of pepsin expression was detected at day 35, followed by a peak of activity at day 40, coinciding with the change from live to artificial food. Totoaba larvae showed a fully morphologically developed digestive system between 24 and 28 dph, as demonstrated by histological observations. However, gene expression and activity of alkaline and acid proteases were detected earlier, indicating the functionality of the exocrine pancreas and stomach before the complete morphological development of the digestive organs. These results showed that integrative studies are needed to fully understand the development of the digestive system from a morphological and functional point of views, since the histological organization of digestive structures does not reflect their real functionality. These results indicate that the digestive system of totoaba develops rapidly during the first days post-hatch, especially for alkaline proteases, and the stomach

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  9. Cytomegalovirus protease targeted prodrug development.

    Science.gov (United States)

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  10. The Arabidopsis Mitochondrial Protease FtSH4 Is Involved in Leaf Senescence via Regulation of WRKY-Dependent Salicylic Acid Accumulation and Signaling.

    Science.gov (United States)

    Zhang, Shengchun; Li, Cui; Wang, Rui; Chen, Yaxue; Shu, Si; Huang, Ruihua; Zhang, Daowei; Li, Jian; Xiao, Shi; Yao, Nan; Yang, Chengwei

    2017-04-01

    Mitochondria and autophagy play important roles in the networks that regulate plant leaf senescence and cell death. However, the molecular mechanisms underlying the interactions between mitochondrial signaling and autophagy are currently not well understood. This study characterized the function of the Arabidopsis ( Arabidopsis thaliana ) mitochondrial AAA-protease gene FtSH4 in regulating autophagy and senescence, finding that FtSH4 mediates WRKY-dependent salicylic acid (SA) accumulation and signaling. Knockout of FtSH4 in the ftsh4-4 mutant resulted in severe leaf senescence, cell death, and high autophagy levels. The level of SA increased dramatically in the ftsh4-4 mutant. Expression of nahG in the ftsh4-4 mutant led to decreased SA levels and suppressed the leaf senescence and cell death phenotypes. The transcript levels of several SA synthesis and signaling genes, including SALICYLIC ACID INDUCTION DEFICIENT2 ( SID2 ), NON-RACE-SPECIFIC DISEASE RESISTANCE1 ( NDR1 ), and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 ( NPR1 ), increased significantly in the ftsh4-4 mutants compared with the wild type. Loss of function of SID2 , NDR1 , or NPR1 in the ftsh4-4 mutant reversed the ftsh4-4 senescence and autophagy phenotypes. Furthermore, ftsh4-4 mutants had elevated levels of transcripts of several WRKY genes, including WRKY40 , WRKY46 , WRKY51 , WRKY60 , WRKY63 , and WRKY75 ; all of these WRKY proteins can bind to the promoter of SID2 Loss of function of WRKY75 in the ftsh4-4 mutants decreased the levels of SA and reversed the senescence phenotype. Taken together, these results suggest that the mitochondrial ATP-dependent protease FtSH4 may regulate the expression of WRKY genes by modifying the level of reactive oxygen species and the WRKY transcription factors that control SA synthesis and signaling in autophagy and senescence. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Eicosapentaenoic acid in cancer improves body composition and modulates metabolism.

    Science.gov (United States)

    Pappalardo, Giulia; Almeida, Ana; Ravasco, Paula

    2015-04-01

    The objective of this review article is to present the most recent intervention studies with EPA on nutritional outcomes in cancer patients, e.g. nutritional status, weight & lean body mass. For this purpose a PubMed(®) and MedLine(®) search of the published literature up to and including January 2014 that contained the keywords: cancer, sarcopenia, EPA, ω-3 fatty acids, weight, intervention trial, muscle mass was conducted. The collected data was summarized and written in text format and in tables that contained: study design, patient' population, sample size, statistical significance and results of the intervention. The paper will cover malignancy, body composition, intervention with EPA, physiological mechanisms of action of EPA, effect of EPA on weight and body composition, future research. In cancer patients deterioration of muscle mass can be present regardless of body weight or Body Mass Index (BMI). Thus, sarcopenia in cancer patients with excessive fat mass (FM), entitled sarcopenic obesity, has gained greater relevance in clinical practice; it can negatively influence patients' functional status, tolerance to treatments & disease prognosis. The search for an effective nutritional intervention that improves body composition (preservation of muscle mass and muscle quality) is of utmost importance for clinicians and patients. The improvement of muscle quality is an even more recent area of interest because it has probable implications in patients' prognosis. Eicosapentaenoic acid (EPA) has been identified as a promising nutrient with the wide clinical benefits. Several mechanisms have been proposed to explain EPA potential benefits on body composition: inhibition of catabolic stimuli by modulating pro-inflammatory cytokines production and enhancing insulin sensitivity that induces protein synthesis; also, EPA may attenuate deterioration of nutritional status resulting from antineoplastic therapies by improving calorie and protein intake as well. Indeed

  12. The bile acid sensor FXR protects against dyslipidemia and aortic plaques development induced by the HIV protease inhibitor ritonavir in mice.

    Directory of Open Access Journals (Sweden)

    Andrea Mencarelli

    Full Text Available BACKGROUND: Although human immunodeficiency virus (HIV-related morbidity and mortality rates in patients treated with a combination of high active antiretroviral therapy (HAART have declined, significant metabolic/vascular adverse effects associated with the long term use of HIV protease inhibitors (PIs have emerged as a significant side effect. Here we illustrate that targeting the bile acid sensor farnesoid X receptor (FXR protects against dyslipidemia and vascular injury induced HIV-PIs in rodents. METHODOLOGY/PRINCIPAL FINDINGS: Administration of the HIV PI ritonavir to wild type mice increased plasma triacylglycerols and cholesterol levels and this effect was exacerbated by dosing ritonavir to mice harbouring a disrupted FXR. Dyslipidemia induced by ritonavir associated with a shift in the liver expression of signature genes, Sterol Regulatory Element-Binding Protein (SREBP-1 and fatty acid synthase. Treating wild type mice with the FXR agonist (chenodeoxycholic acid, CDCA protected against development of dyslipidemia induced by ritonavir. Administration of ritonavir to ApoE(-/- mice, a strain that develop spontaneously atherosclerosis, increased the extent of aortic plaques without worsening the dyslipidemia. Treating these mice with CDCA reduced the extent of aortic plaques by 70% without changing plasma lipoproteins or the liver expression of signature genes. A beneficial effect on aortic plaques was also obtained by treating ApoE(-/- mice with gemfibrozil, a PPARα agonist. FXR activation counter-regulated induction of expression/activity of CD36 caused by HIV-PIs in circulating monocytes and aortic plaques. In macrophages cell lines, CDCA attenuated CD36 induction and uptake of acetylated LDL caused by ritonavir. Natural and synthetic FXR ligands reduced the nuclear translocation of SREBP1c caused by ritonavir. CONCLUSIONS/SIGNIFICANCE: Activation of the bile acid sensor FXR protects against dyslipidemia and atherosclerotic caused by

  13. Microbial alkaline proteases: Optimization of production parameters and their properties

    Directory of Open Access Journals (Sweden)

    Kanupriya Miglani Sharma

    2017-06-01

    Full Text Available Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.

  14. Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

    Science.gov (United States)

    Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-15

    The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors

  15. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza

    2015-06-01

    Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

  16. The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

    Directory of Open Access Journals (Sweden)

    Mark J White

    2011-03-01

    Full Text Available Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

  17. AtWRKY22 promotes susceptibility to aphids and modulates salicylic acid and jasmonic acid signalling.

    Science.gov (United States)

    Kloth, Karen J; Wiegers, Gerrie L; Busscher-Lange, Jacqueline; van Haarst, Jan C; Kruijer, Willem; Bouwmeester, Harro J; Dicke, Marcel; Jongsma, Maarten A

    2016-05-01

    Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA-SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  18. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  19. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    International Nuclear Information System (INIS)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-01-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  20. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

    Science.gov (United States)

    Stransky, Laura A.; Forgac, Michael

    2015-01-01

    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  1. Understanding serine proteases implications on Leishmania spp lifecycle.

    Science.gov (United States)

    Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

    2018-01-01

    Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference...

  3. Combined amino acids modulation with H 2 O 2 stress for ...

    African Journals Online (AJOL)

    Strategies of amino acids addition coupled with H2O2 stresses were developed for glutathione (GSH) overproduction in high cell density (HCD) cultivation of Candida utilis. Based on the fact that glycine shows two functions of promoting cells growth as well as GSH production, precursor amino acids modulations of feeding ...

  4. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

  5. Does Branched-Chain Amino Acids Supplementation Modulate Skeletal Muscle Remodeling through Inflammation Modulation? Possible Mechanisms of Action

    Directory of Open Access Journals (Sweden)

    Humberto Nicastro

    2012-01-01

    Full Text Available Skeletal muscle protein turnover is modulated by intracellular signaling pathways involved in protein synthesis, degradation, and inflammation. The proinflammatory status of muscle cells, observed in pathological conditions such as cancer, aging, and sepsis, can directly modulate protein translation initiation and muscle proteolysis, contributing to negative protein turnover. In this context, branched-chain amino acids (BCAAs, especially leucine, have been described as a strong nutritional stimulus able to enhance protein translation initiation and attenuate proteolysis. Furthermore, under inflammatory conditions, BCAA can be transaminated to glutamate in order to increase glutamine synthesis, which is a substrate highly consumed by inflammatory cells such as macrophages. The present paper describes the role of inflammation on muscle remodeling and the possible metabolic and cellular effects of BCAA supplementation in the modulation of inflammatory status of skeletal muscle and the consequences on protein synthesis and degradation.

  6. Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

    Science.gov (United States)

    Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

    2017-11-29

    Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

  7. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

    Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

  8. Indispensable Role of Proteases in Plant Innate Immunity.

    Science.gov (United States)

    Balakireva, Anastasia V; Zamyatnin, Andrey A

    2018-02-23

    Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

  9. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  10. Modulation of folate production in lactic acid bacteria

    NARCIS (Netherlands)

    Wegkamp, H.B.A.

    2008-01-01

    Food fortification has proven to be very useful in reducing health problems associated with mal-intake of essential nutrients, such as the B-vitamin folate. Folate is used as one-carbon donor/acceptor in several biochemical processes like synthesis of DNA, RNA and some amino acids. Sufficient intake

  11. Guiding principle for crystalline Si photovoltaic modules with high tolerance to acetic acid

    Science.gov (United States)

    Masuda, Atsushi; Hara, Yukiko

    2018-04-01

    A guiding principle for highly reliable crystalline Si photovoltaic modules, especially those with high tolerance to acetic acid generated by hydrolysis reaction between water vapor and an ethylene-vinyl acetate (EVA) encapsulant, is proposed. Degradation behavior evaluated by the damp heat test strongly depends on Ag finger electrodes and also EVA encapsulants. The acetic acid concentration in EVA on the glass side directly determines the degradation behavior. The most important factor for high tolerance is the type of Ag finger electrode materials when using an EVA encapsulant. Photovoltaic modules using newly developed crystalline Si cells with improved Ag finger electrode materials keep their maximum power of 80% of the initial value even after the damp heat test at 85 °C and 85% relative humidity for 10000 h. The pattern of dark regions in electroluminescence images is also discussed on the basis of the dynamics of acetic acid in the modules.

  12. Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes

    NARCIS (Netherlands)

    Punt, P.J.; Schuren, F.H.J.; Lehmbeck, J.; Christensen, T.; Hjort, C.; Hondel, C.A.M.J.J. van den

    2008-01-01

    Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A.,

  13. Modulation of hepatic steatosis by dietary fatty acids.

    Science.gov (United States)

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-02-21

    Non-alcoholic fatty liver disease (NAFLD) describes a range of conditions caused by fat deposition within liver cells. Liver fat content reflects the equilibrium between several metabolic pathways involved in triglyceride synthesis and disposal, such as lipolysis in adipose tissue and de novo lipogenesis, triglyceride esterification, fatty acid oxidation and very-low-density lipoprotein synthesis/secretion in hepatic tissue. In particular, it has been demonstrated that hepatic de novo lipogenesis plays a significant role in NAFLD pathogenesis. It is widely known that the fatty acid composition of the diet influences hepatic lipogenesis along with other metabolic pathways. Therefore, dietary fat may not only be involved in the pathogenesis of hepatic steatosis, but may also prevent and/or reverse hepatic fat accumulation. In this review, major data from the literature about the role of some dietary fats as a potential cause of hepatic fat accumulation or as a potential treatment for NAFLD are described. Moreover, biochemical mechanisms responsible for an increase or decrease in hepatic lipid content are critically analyzed. It is noteworthy that both quantitative and qualitative aspects of dietary fat influence triglyceride deposition in the liver. A high-fat diet or the dietary administration of conjugated linoleic acids induced hepatic steatosis. In contrast, supplementation of the diet with krill oil or pine nut oil helped in the prevention and/or in the treatment of steatotic liver. Quite interesting is the "case" of olive oil, since several studies have often provided different and/or conflicting results in animal models.

  14. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

  15. Modulation of hepatic steatosis by dietary fatty acids

    Science.gov (United States)

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD) describes a range of conditions caused by fat deposition within liver cells. Liver fat content reflects the equilibrium between several metabolic pathways involved in triglyceride synthesis and disposal, such as lipolysis in adipose tissue and de novo lipogenesis, triglyceride esterification, fatty acid oxidation and very-low-density lipoprotein synthesis/secretion in hepatic tissue. In particular, it has been demonstrated that hepatic de novo lipogenesis plays a significant role in NAFLD pathogenesis. It is widely known that the fatty acid composition of the diet influences hepatic lipogenesis along with other metabolic pathways. Therefore, dietary fat may not only be involved in the pathogenesis of hepatic steatosis, but may also prevent and/or reverse hepatic fat accumulation. In this review, major data from the literature about the role of some dietary fats as a potential cause of hepatic fat accumulation or as a potential treatment for NAFLD are described. Moreover, biochemical mechanisms responsible for an increase or decrease in hepatic lipid content are critically analyzed. It is noteworthy that both quantitative and qualitative aspects of dietary fat influence triglyceride deposition in the liver. A high-fat diet or the dietary administration of conjugated linoleic acids induced hepatic steatosis. In contrast, supplementation of the diet with krill oil or pine nut oil helped in the prevention and/or in the treatment of steatotic liver. Quite interesting is the “case” of olive oil, since several studies have often provided different and⁄or conflicting results in animal models. PMID:24587652

  16. Biophysical insight into structure-function relation of Allium sativum Protease Inhibitor by thermal, chemical and pH-induced modulation using comprehensive spectroscopic analysis.

    Science.gov (United States)

    Shamsi, Tooba Naz; Parveen, Romana; Naz, Huma; Haque, Md Anzarul; Fatima, Sadaf

    2017-10-01

    In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of ∼15kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+β protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70°C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. T m (midpoint of denaturation), ΔC p (constant pressure heat capacity change) and ΔH m (van't Hoff enthalpy change at T m were calculated to be 41.25±0.2°C, 1.3±0.07kcalmol -1 K -1 and 61±2kcalmol -1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Lon protease modulates virulence traits in Erwinia amylovora by direct monitoring of major regulators and indirectly through the Rcs and Gac-Csr regulatory systems.

    Science.gov (United States)

    Lee, Jae Hoon; Ancona, Veronica; Zhao, Youfu

    2018-04-01

    Lon, an ATP-dependent protease in bacteria, influences diverse cellular processes by degrading damaged, misfolded and short-lived regulatory proteins. In this study, we characterized the effects of lon mutation and determined the molecular mechanisms underlying Lon-mediated virulence regulation in Erwinia amylovora, an enterobacterial pathogen of apple. Erwinia amylovora depends on the type III secretion system (T3SS) and the exopolysaccharide (EPS) amylovoran to cause disease. Our results showed that mutation of the lon gene led to the overproduction of amylovoran, increased T3SS gene expression and the non-motile phenotype. Western blot analyses showed that mutation in lon directly affected the accumulation and stability of HrpS/HrpA and RcsA. Mutation in lon also indirectly influenced the expression of flhD, hrpS and csrB through the accumulation of the RcsA/RcsB proteins, which bind to the promoter of these genes. In addition, lon expression is under the control of CsrA, possibly at both the transcriptional and post-transcriptional levels. Although mutation in csrA abolished both T3SS and amylovoran production, deletion of the lon gene in the csrA mutant only rescued amylovoran production, but not T3SS. These results suggest that CsrA might positively control both T3SS and amylovoran production partly by suppressing Lon, whereas CsrA may also play a critical role in T3SS by affecting unknown targets. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  18. Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

    Science.gov (United States)

    Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

    2000-09-01

    Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

  19. A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid.

    Science.gov (United States)

    Castro, Maite A; Beltrán, Felipe A; Brauchi, Sebastián; Concha, Ilona I

    2009-07-01

    In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.

  20. Uncovering co-expression gene network modules regulating fruit acidity in diverse apples.

    Science.gov (United States)

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Zhong, Gan-Yuan; Xu, Kenong

    2015-08-16

    Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background.

  1. Modulation of i-motif thermodynamic stability by the introduction of UNA (unlocked nucleic acid) monomers

    DEFF Research Database (Denmark)

    Pasternak, Anna; Wengel, Jesper

    2011-01-01

    The influence of acyclic RNA derivatives, UNA (unlocked nucleic acid) monomers, on i-DNA thermodynamic stability has been investigated. The 22 nt human telomeric fragment was chosen as the model sequence for stability studies. UNA monomers modulate i-motif stability in a position-depending manner...

  2. Abscisic Acid-Cytokinin Antagonism Modulates Resistance Against Pseudomonas syringae in Tobacco.

    Science.gov (United States)

    Großkinsky, Dominik K; van der Graaff, Eric; Roitsch, Thomas

    2014-12-01

    Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohormones have been demonstrated to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco, antagonistic interaction of these phytohormones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based on these results, we conclude that reduction of abscisic acid levels by enhanced abscisic acid catabolism strongly contributes to cytokinin-mediated resistance effects. Thus, the identified cytokinin-abscisic acid antagonism is a novel regulatory mechanism in plant immunity.

  3. Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.

    Science.gov (United States)

    Neustadt, Madlen; Costina, Victor; Kupfahl, Claudio; Buchheidt, Dieter; Eckerskorn, Christoph; Neumaier, Michael; Findeisen, Peter

    2009-06-01

    Early diagnosis of life-threatening invasive aspergillosis in neutropenic patients remains challenging because current laboratory methods have limited diagnostic sensitivity and/or specificity. Aspergillus species are known to secrete various pathogenetically relevant proteases and the monitoring of their protease activity in serum specimens might serve as a new diagnostic approach.For the characterization and identification of secreted proteases, the culture supernatant of Aspergillus fumigatus was fractionated using free flow electrophoresis (Becton Dickinson). Protease activity of separated fractions was measured using fluorescently labeled reporter peptides. Fractions were also co-incubated in parallel with various protease inhibitors that specifically inhibit a distinct class of proteases e.g. metallo- or cysteine-proteases. Those fractions with high protease activity were further subjected to LC-MS/MS analysis for protease identification. The highest protease activity was measured in fractions with an acidic pH range. The results of the 'inhibitor-panel' gave a clear indication that it is mainly metallo- and serine-proteases that are involved in the degradation of reporter peptides. Furthermore, several proteases were identified that facilitate the optimization of reporter peptides for functional protease profiling as a diagnostic tool for invasive aspergillosis.

  4. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

    Full Text Available 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. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

  5. Discovery of novel histidine-derived lipo-amino acids: applied in the synthesis of ultra-short antimicrobial peptidomimetics having potent antimicrobial activity, salt resistance and protease stability.

    Science.gov (United States)

    Ahn, Mija; Murugan, Ravichandran N; Jacob, Binu; Hyun, Jae-Kyung; Cheong, Chaejoon; Hwang, Eunha; Park, Hyo-Nam; Seo, Ji-Hyung; Srinivasrao, G; Lee, Kyung S; Shin, Song Yub; Bang, Jeong Kyu

    2013-10-01

    Here we report for the first time the synthesis of Histidine (His) derived lipo-amino acids having pendant lipid tails at N(τ)- and N(π)-positions on imidazole group of His and applied it into synthesis of lipo-peptides. The attachment of His-derived lipo-amino acid into the very short inactive cationic peptides endows potent antimicrobial activity against Gram-positive and Gram-negative bacteria without hemolytic activity. Furthermore, our designed His-derived lipo-peptidomimetics (HDLPs) consisting of two or three residues displayed strong anti-MRSA activity and protease stability as well as retained potent antimicrobial activity under high salt concentration. Our results demonstrate that the novel lipo-amino acid is highly flexible to synthesize and carry out the extensive structure-activity relationship (SAR) on lipo-antimicrobial peptidomimetics and represents a unique amenable platform for modifying parameters important for antimicrobial activity. Through this study, we proved that the discovery of His-derived lipo-amino acid and the corresponding HDLPs are an excellent candidate as a lead compound for the development of novel antimicrobial agents. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  6. Abscisic acid-cytokinin antagonism modulates resistance against pseudomonas syringae in Tobacco

    DEFF Research Database (Denmark)

    Grosskinsky, Dominik Kilian; van der Graaff, Eric; Roitsch, Thomas Georg

    2014-01-01

    Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohormones have been demonstrated to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant...... immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction...... of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco...

  7. Refractive index modulation in polymer film doped with diazo Meldrum's acid

    Science.gov (United States)

    Zanutta, Alessio; Villa, Filippo; Bertarelli, Chiara; Bianco, Andrea

    2016-08-01

    Diazo Meldrum's acid undergoes a photoreaction induced by UV light and it is used as photosensitizer in photoresists. Upon photoreaction, a change in refractive index occurs, which makes this system interesting for volume holography. We report on the sublimation effect at room temperature and the effect of photoirradiation on the refractive index in thin films of CAB (Cellulose acetate butyrate) doped with different amount of diazo Meldrum's acid. A net modulation of the refractive index of 0.01 is achieved with 40% of doping ratio together with a reduction of the film thickness.

  8. Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes

    NARCIS (Netherlands)

    Gustafson, LA; Neeft, M; Reijngoud, DJ; Kuipers, F; Sauerwein, HP; Romijn, JA; Herling, AW; Burger, HJ; Meijer, AJ

    2001-01-01

    We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also

  9. Fatty Acid Modulation of the Endocannabinoid System and the Effect on Food Intake and Metabolism

    Directory of Open Access Journals (Sweden)

    Shaan S. Naughton

    2013-01-01

    Full Text Available Endocannabinoids and their G-protein coupled receptors (GPCR are a current research focus in the area of obesity due to the system’s role in food intake and glucose and lipid metabolism. Importantly, overweight and obese individuals often have higher circulating levels of the arachidonic acid-derived endocannabinoids anandamide (AEA and 2-arachidonoyl glycerol (2-AG and an altered pattern of receptor expression. Consequently, this leads to an increase in orexigenic stimuli, changes in fatty acid synthesis, insulin sensitivity, and glucose utilisation, with preferential energy storage in adipose tissue. As endocannabinoids are products of dietary fats, modification of dietary intake may modulate their levels, with eicosapentaenoic and docosahexaenoic acid based endocannabinoids being able to displace arachidonic acid from cell membranes, reducing AEA and 2-AG production. Similarly, oleoyl ethanolamide, a product of oleic acid, induces satiety, decreases circulating fatty acid concentrations, increases the capacity for β-oxidation, and is capable of inhibiting the action of AEA and 2-AG in adipose tissue. Thus, understanding how dietary fats alter endocannabinoid system activity is a pertinent area of research due to public health messages promoting a shift towards plant-derived fats, which are rich sources of AEA and 2-AG precursor fatty acids, possibly encouraging excessive energy intake and weight gain.

  10. THE COMBINATION OF α-LIPOIC ACID INTAKE WITH ECCENTRIC EXERCISE MODULATES ERYTHROPOIETIN RELEASE

    Directory of Open Access Journals (Sweden)

    B. Morawin

    2014-08-01

    Full Text Available The generation of reactive nitrogen/oxygen species (RN/OS represents an important mechanism in erythropoietin (EPO expression and skeletal muscle adaptation to physical and metabolic stress. RN/OS generation can be modulated by intense exercise and nutrition supplements such as α-lipoic acid, which demonstrates both anti- and pro-oxidative action. The study was designed to show the changes in the haematological response through the combination of α-lipoic acid intake with running eccentric exercise. Sixteen healthy young males participated in the randomised and placebo-controlled study. The exercise trial involved a 90-min run followed by a 15-min eccentric phase at 65% VO2max (-10% gradient. It significantly increased serum concentrations of nitric oxide (NO, hydrogen peroxide (H2O2 and pro-oxidative products such as 8-isoprostanes (8-iso, lipid peroxides (LPO and protein carbonyls (PC. α-Lipoic acid intake (Thiogamma: 1200 mg daily for 10 days prior to exercise resulted in a 2-fold elevation of serum H2O2 concentration before exercise, but it prevented the generation of NO, 8-iso, LPO and PC at 20 min, 24 h, and 48 h after exercise. α-Lipoic acid also elevated serum EPO level, which highly correlated with NO/H2O2 ratio (r=0.718, P<0.01. Serum total creatine kinase (CK activity, as a marker of muscle damage, reached a peak at 24 h after exercise (placebo 732 ± 207 IU · L-1, α-lipoic acid 481 ± 103 IU · L-1, and correlated with EPO (r = 0.478, P<0.01 in the α-lipoic acid group. In conclusion, the intake of high α-lipoic acid modulates RN/OS generation, enhances EPO release and reduces muscle damage after running eccentric exercise.

  11. Characterization of a membrane-associated serine protease in Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.; St John, A.C.

    1987-01-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

  12. Multifunctional Mitochondrial AAA Proteases.

    Science.gov (United States)

    Glynn, Steven E

    2017-01-01

    Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

  13. Modulating the Global Response Regulator, LuxO of V. cholerae Quorum Sensing System Using a Pyrazine Dicarboxylic Acid Derivative (PDCApy: An Antivirulence Approach

    Directory of Open Access Journals (Sweden)

    M. Hema

    2017-10-01

    Full Text Available Vibrio cholerae is a Gram-negative pathogen which causes acute diarrhoeal disease, cholera by the expression of virulence genes through quorum sensing (QS mechanism. The QS circuit of V. cholerae is controlled by the global quorum regulator, LuxO, which at low cell density (LCD state produces major virulence factors such as, toxin co-regulated pilus (TCP and cholera toxin (CT to mediate infection. On the contrary, at the high cell density (HCD state the virulent genes are downregulated and the vibrios are detached from the host intestinal epithelial cells, promoted by HapA protease. Hence, targeting the global regulator LuxO would be a promising approach to modulate the QS to curtail V. cholerae pathogenesis. In our earlier studies, LuxO targeted ligand, 2,3 pyrazine dicarboxylic acid (PDCA and its derivatives having desired pharmacophore properties were chemically synthesized and were shown to have biofilm inhibition as well as synergistic activity with the conventionally used antibiotics. In the present study, the QS modulatory effect of the PDCA derivative with pyrrolidine moiety designated as PDCApy against the V. cholerae virulence gene expression was analyzed at various growth phases. The data significantly showed a several fold reduction in the expression of the genes, tcp and ct whereas the expression of hapR was upregulated at the LCD state. In addition, PDCApy reduced the adhesion and invasion of the vibrios onto the INT407 intestinal cell lines. Collectively, our data suggest that PDCApy could be a potential QS modulator (QSM for the antivirulence therapeutic approach.

  14. Synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid vinyl-ACCA) derivatives: key intermediates for the preparation of inhibitors of the hepatitis C virus NS3 protease.

    Science.gov (United States)

    Beaulieu, Pierre L; Gillard, James; Bailey, Murray D; Boucher, Colette; Duceppe, Jean-Simon; Simoneau, Bruno; Wang, Xiao-Jun; Zhang, Li; Grozinger, Karl; Houpis, Ioannis; Farina, Vittorio; Heimroth, Heidi; Krueger, Thomas; Schnaubelt, Jürgen

    2005-07-22

    (1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) is a key building block in the synthesis of potent inhibitors of the hepatitis C virus NS3 protease such as BILN 2061, which was recently shown to dramatically reduce viral load after administration to patients infected with HCV genotype 1. We have developed a scalable process that delivers derivatives of this unusual amino acid in >99% ee. The strategy was based on the dialkylation of a glycine Schiff base using trans-1,4-dibromo-2-butene as an electrophile to produce racemic vinyl-ACCA, which was subsequently resolved using a readily available, inexpensive esterase enzyme (Alcalase 2.4L). Factors that affect diastereoselection in the initial dialkylation steps were examined and the conditions optimized to deliver the desired diastereomer selectively. Product inhibition, which was encountered during the enzymatic resolution step, initially resulted in prolonged cycle times. Enrichment of racemic vinyl-ACCA through a chemical resolution via diastereomeric salt formation or the use of forcing conditions in the enzymatic reaction both led to improvements in throughput and the development of a viable process. The chemistry described herein was scaled up to produce multikilogram quantities of this building block.

  15. Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice

    International Nuclear Information System (INIS)

    Zhang, Youcai; Limaye, Pallavi B.; Renaud, Helen J.; Klaassen, Curtis D.

    2014-01-01

    Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin + imipenem and cephalothin + neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin + imipenem but stimulated by cephalothin + neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism. - Highlights: • Various antibiotics have different effects on intestinal bacteria. • Antibiotics alter bile acid composition in mouse liver and intestine. • Antibiotics influence genes involved in bile acid homeostasis. • Clostridia appear to be important for secondary bile acid formation

  16. Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youcai; Limaye, Pallavi B.; Renaud, Helen J.; Klaassen, Curtis D., E-mail: curtisklaassenphd@gmail.com

    2014-06-01

    Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin + imipenem and cephalothin + neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin + imipenem but stimulated by cephalothin + neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism. - Highlights: • Various antibiotics have different effects on intestinal bacteria. • Antibiotics alter bile acid composition in mouse liver and intestine. • Antibiotics influence genes involved in bile acid homeostasis. • Clostridia appear to be important for secondary bile acid formation.

  17. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

  18. How Does Amino Acid Ligand Modulate Au Core Structure and Characteristics in Peptide Coated Au Nanocluster?

    Science.gov (United States)

    Li, Nan; Li, Xu; Zhao, Hongkang; Zhao, Lina

    2018-03-01

    The atomic structures and the corresponding physicochemical properties of peptide coated Au nanoclusters determine their distinctive biological targeting applications. To learn the modulation of amino acid ligand on the atomic structure and electronic characteristics of coated Au core is the fundamental knowledge for peptide coated Au nanocluster design and construction. Based on our recent coated Au nanocluster configuration study (Nanoscale, 2016, 8, 11454), we built the typically simplified Au13(Cys-Au-Cys) system to more clearly learn the basic modulation information of amino acid ligand on Au core by the density functional theory (DFT) calculations. There are two isomers as ligand adjacent bonding (Iso1) and diagonal bonding (Iso2) to Au13 cores. The geometry optimizations indicate the adjacent bonding Iso1 is more stable than Iso2. More important, the Au13 core of Iso1 distorts much more significantly than that of Iso2 by Cys-Au-Cys bonding through the root-mean-square deviation (RMSD) analysis, which modulate their electronic characteristics in different ways. In addition, the frontier molecular orbital results of Au13(Cys-Au-Cys) isomers confirm that the Au cores mainly determine the blue shifts of Au13(Cys-Au-Cys) systems versus the original Au13 core in their UV-visible absorption spectrum studies. The configuration of Au13 core performs deformation under Cys-Au-Cys ligand modulation to reach new stability with distinct atomic structure and electronic properties, which could be the theory basis for peptide coated AuNCs design and construction.

  19. The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium

    International Nuclear Information System (INIS)

    Ferain, Aline; Bonnineau, Chloé; Neefs, Ineke; Rees, Jean François; Larondelle, Yvan; Schamphelaere, Karel A.C.De; Debier, Cathy

    2016-01-01

    Highlights: • The phospholipid composition of rainbow trout liver cells was successfully changed. • Cell phospholipids influenced methylmercury (MeHg) and cadmium (Cd) toxicity. • Cells enriched in 18:3n-3, 20:5n-3 or 22:5n-6 were more resistant to MeHg and Cd. • Cell enrichment in 22:6n-3 increased resistance to Cd but not MeHg. - Abstract: The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium. Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enriching the growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i) the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly

  20. The fatty acid profile of rainbow trout liver cells modulates their tolerance to methylmercury and cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Ferain, Aline, E-mail: aline.ferain@uclouvain.be [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Bonnineau, Chloé [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Irstea, UR MALY, Centre de Lyon-Villeurbanne, rue de la Doua 5/32108, F-69616 Villeurbanne (France); Neefs, Ineke; Rees, Jean François; Larondelle, Yvan [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium); Schamphelaere, Karel A.C.De [Laboratory of Environmental Toxicology and Aquatic Ecology, Environmental Toxicology Unit, Ghent University, J. Plateaustraat 22, B-9000 Ghent (Belgium); Debier, Cathy [Institute of Life Sciences, Université catholique de Louvain, Place Croix du Sud 2/L7.05.08, B-1348 Louvain-la-Neuve (Belgium)

    2016-08-15

    Highlights: • The phospholipid composition of rainbow trout liver cells was successfully changed. • Cell phospholipids influenced methylmercury (MeHg) and cadmium (Cd) toxicity. • Cells enriched in 18:3n-3, 20:5n-3 or 22:5n-6 were more resistant to MeHg and Cd. • Cell enrichment in 22:6n-3 increased resistance to Cd but not MeHg. - Abstract: The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium. Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enriching the growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i) the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly

  1. Variable context Markov chains for HIV protease cleavage site prediction.

    Science.gov (United States)

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

  2. Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines.

    NARCIS (Netherlands)

    Dube, N.; Delvin, E.; Yotov, W.; Garofalo, C.; Bendayan, M.; Veerkamp, J.H.; Levy, E.

    2001-01-01

    Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic

  3. Citric acid induced W18O49 electrochromic films with enhanced optical modulation

    Science.gov (United States)

    Xie, Junliang; Song, Bin; Zhao, Gaoling; Han, Gaorong

    2018-06-01

    Electrochromic materials exhibit promising applications in energy-saving fields for their ability to control heat from outdoors. Nanostructured W18O49 has drawn attention for its one-dimensional structure to transfer charge efficiently as a remarkable electrochromic material. W18O49 bi-layer films were fabricated through a facile one-step solvothermal process with citric acid as a chelating agent. The addition of citric acid improved the deposition on the substance, and a nanostructured film with a denser layer at the bottom and a tussock-like upper layer was obtained. The bi-layer film exhibited an enhanced optical modulation of 68.7%, a coloration efficiency of 82.1 cm2/C with stability over 400 cycles, and fast response times (1.4 s and 2.3 s for bleaching and coloring), with expectation to be applied in the electrochromic field.

  4. GID1 modulates stomatal response and submergence tolerance involving abscisic acid and gibberellic acid signaling in rice.

    Science.gov (United States)

    Du, Hao; Chang, Yu; Huang, Fei; Xiong, Lizhong

    2015-11-01

    Plant responses to abiotic stresses are coordinated by arrays of growth and developmental programs. Gibberellic acid (GA) and abscisic acid (ABA) play critical roles in the developmental programs and environmental responses, respectively, through complex signaling and metabolism networks. However, crosstalk between the two phytohormones in stress responses remains largely unknown. In this study, we report that GIBBERELLIN-INSENSITIVE DWARF 1 (GID1), a soluble receptor for GA, regulates stomatal development and patterning in rice (Oryza sativa L.). The gid1 mutant showed impaired biosynthesis of endogenous ABA under drought stress conditions, but it exhibited enhanced sensitivity to exogenous ABA. Scanning electron microscope and infrared thermal image analysis indicated an increase in the stomatal conductance in the gid1 mutant under drought conditions. Interestingly, the gid1 mutant had increased levels of chlorophyll and carbohydrates under submergence conditions, and showed enhanced reactive oxygen species (ROS)-scavenging ability and submergence tolerance compared with the wild-type. Further analyses suggested that the function of GID1 in submergence responses is partially dependent on ABA, and GA signaling by GID1 is involved in submergence tolerance by modulating carbohydrate consumption. Taken together, these findings suggest GID1 plays distinct roles in stomatal response and submergence tolerance through both the ABA and GA signaling pathways in rice. © 2014 Institute of Botany, Chinese Academy of Sciences.

  5. Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology.

    Science.gov (United States)

    Khadge, Saraswoti; Sharp, John Graham; Thiele, Geoffrey M; McGuire, Timothy R; Klassen, Lynell W; Duryee, Michael J; Britton, Holly C; Dafferner, Alicia J; Beck, Jordan; Black, Paul N; DiRusso, Concetta C; Talmadge, James

    2018-02-01

    Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. ERECTA, salicylic acid, abscisic acid, and jasmonic acid modulate quantitative disease resistance of Arabidopsis thaliana to Verticillium longisporum.

    Science.gov (United States)

    Häffner, Eva; Karlovsky, Petr; Splivallo, Richard; Traczewska, Anna; Diederichsen, Elke

    2014-04-01

    Verticillium longisporum is a soil-borne vascular pathogen infecting cruciferous hosts such as oilseed rape. Quantitative disease resistance (QDR) is the major control means, but its molecular basis is poorly understood so far. Quantitative trait locus (QTL) mapping was performed using a new (Bur×Ler) recombinant inbred line (RIL) population of Arabidopsis thaliana. Phytohormone measurements and analyses in defined mutants and near-isogenic lines (NILs) were used to identify genes and signalling pathways that underlie different resistance QTL. QTL for resistance to V. longisporum-induced stunting, systemic colonization by the fungus and for V. longisporum-induced chlorosis were identified. Stunting resistance QTL were contributed by both parents. The strongest stunting resistance QTL was shown to be identical with Erecta. A functional Erecta pathway, which was present in Bur, conferred partial resistance to V. longisporum-induced stunting. Bur showed severe stunting susceptibility in winter. Three stunting resistance QTL of Ler origin, two co-localising with wall-associated kinase-like (Wakl)-genes, were detected in winter. Furthermore, Bur showed a much stronger induction of salicylic acid (SA) by V. longisporum than Ler. Systemic colonization was controlled independently of stunting. The vec1 QTL on chromosome 2 had the strongest effect on systemic colonization. The same chromosomal region controlled the level of abscisic acid (ABA) and jasmonic acid (JA) in response to V. longisporum: The level of ABA was higher in colonization-susceptible Ler than in colonization-resistant Bur after V. longisporum infection. JA was down-regulated in Bur after infection, but not in Ler. These differences were also demonstrated in NILs, varying only in the region containing vec1. All phytohormone responses were shown to be independent of Erecta. Signalling systems with a hitherto unknown role in the QDR of A. thaliana against V. longisporum were identified: Erecta mediated

  7. Gut microbiota alterations and dietary modulation in childhood malnutrition - The role of short chain fatty acids.

    Science.gov (United States)

    Pekmez, Ceyda Tugba; Dragsted, Lars Ove; Brahe, Lena Kirchner

    2018-02-17

    The gut microbiome affects the health status of the host through different mechanisms and is associated with a wide variety of diseases. Both childhood undernutrition and obesity are linked to alterations in composition and functionality of the gut microbiome. One of the possible mechanisms underlying the interplay between microbiota and host metabolism is through appetite-regulating hormones (including leptin, ghrelin, glucagon-like peptide-1). Short chain fatty acids, the end product of bacterial fermentation of non-digestible carbohydrates, might be able to alter energy harvest and metabolism through enteroendocrine cell signaling, adipogenesis and insulin-like growth factor-1 production. Elucidating these mechanisms may lead to development of new modulation practices of the gut microbiota as a potential prevention and treatment strategy for childhood malnutrition. The present overview will briefly outline the gut microbiota development in the early life, gut microbiota alterations in childhood undernutrition and obesity, and whether this relationship is causal. Further we will discuss possible underlying mechanisms in relation to the gut-brain axis and short chain fatty acids, and the potential of probiotics, prebiotics and synbiotics for modulating the gut microbiota during childhood as a prevention and treatment strategy against undernutrition and obesity. Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  8. Efficient modulation of γ-aminobutyric acid type A receptors by piperine derivatives.

    Science.gov (United States)

    Schöffmann, Angela; Wimmer, Laurin; Goldmann, Daria; Khom, Sophia; Hintersteiner, Juliane; Baburin, Igor; Schwarz, Thomas; Hintersteininger, Michael; Pakfeifer, Peter; Oufir, Mouhssin; Hamburger, Matthias; Erker, Thomas; Ecker, Gerhard F; Mihovilovic, Marko D; Hering, Steffen

    2014-07-10

    Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode voltage-clamp technique. GABAAR were expressed in Xenopus laevis oocytes. Structure-activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-induced chloride current modulation (IGABA-max = 1673% ± 146%, EC50 = 51.7 ± 9.5 μM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC50 = 13.8 ± 1.8 μM, IGABA-max = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABAAR modulators.

  9. Synthesis of glycinamides using protease immobilized magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Abha Sahu

    2016-12-01

    Full Text Available In the present investigation, Bacillus subtilis was isolated from slaughterhouse waste and screened for the production of protease enzyme. The purified protease was successfully immobilized on magnetic nanoparticles (MNPs and used for the synthesis of series of glycinamides. The binding and thermal stability of protease on MNPs was confirmed by FTIR spectroscopy and TGA analysis. The surface morphology of MNPs before and after protease immobilization was carried out using SEM analysis. XRD pattern revealed no phase change in MNPs after enzyme immobilization. The processing parameters for glycinamides synthesis viz. temperature, pH, and time were optimized using Response Surface Methodology (RSM by using Design Expert (9.0.6.2. The maximum yield of various amides 2 butyramidoacetic acid (AMD-1,83.4%, 2-benzamidoacetic acid (AMD-2,80.5% and 2,2′((carboxymethyl amino-2-oxoethyl-2-hydroxysuccinylbis(azanediyldiacetic acid (AMD-3,80.8% formed was observed at pH-8, 50 °C and 30 min. The synthesized immobilized protease retained 70% of the initial activity even after 8 cycles of reuse.

  10. Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

    Science.gov (United States)

    Chinnadurai, Gandhi Shree; Krishnan, Sivakumar; Perumal, Palani

    2018-02-01

    The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants

  11. THE COMBINATION OF α-LIPOIC ACID INTAKE WITH ECCENTRIC EXERCISE MODULATES ERYTHROPOIETIN RELEASE.

    Science.gov (United States)

    Morawin, B; Turowski, D; Naczk, M; Siatkowski, I; Zembron-Lacny, A

    2014-08-01

    The generation of reactive nitrogen/oxygen species (RN/OS) represents an important mechanism in erythropoietin (EPO) expression and skeletal muscle adaptation to physical and metabolic stress. RN/OS generation can be modulated by intense exercise and nutrition supplements such as α-lipoic acid, which demonstrates both anti- and pro-oxidative action. The study was designed to show the changes in the haematological response through the combination of α-lipoic acid intake with running eccentric exercise. Sixteen healthy young males participated in the randomised and placebo-controlled study. The exercise trial involved a 90-min run followed by a 15-min eccentric phase at 65% VO2max (-10% gradient). It significantly increased serum concentrations of nitric oxide (NO), hydrogen peroxide (H2O2) and pro-oxidative products such as 8-isoprostanes (8-iso), lipid peroxides (LPO) and protein carbonyls (PC). α-Lipoic acid intake (Thiogamma: 1200 mg daily for 10 days prior to exercise) resulted in a 2-fold elevation of serum H2O2 concentration before exercise, but it prevented the generation of NO, 8-iso, LPO and PC at 20 min, 24 h, and 48 h after exercise. α-Lipoic acid also elevated serum EPO level, which highly correlated with NO/H2O2 ratio (r = 0.718, P exercise (placebo 732 ± 207 IU · L(-1), α-lipoic acid 481 ± 103 IU · L(-1)), and correlated with EPO (r = 0.478, P release and reduces muscle damage after running eccentric exercise.

  12. Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography.

    Science.gov (United States)

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-11-18

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

  13. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Directory of Open Access Journals (Sweden)

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  14. AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling.

    Science.gov (United States)

    Shim, Jae Sung; Jung, Choonkyun; Lee, Sangjoon; Min, Kyunghun; Lee, Yin-Won; Choi, Yeonhee; Lee, Jong Seob; Song, Jong Tae; Kim, Ju-Kon; Choi, Yang Do

    2013-02-01

    The role of AtMYB44, an R2R3 MYB transcription factor, in signaling mediated by jasmonic acid (JA) and salicylic acid (SA) is examined. AtMYB44 is induced by JA through CORONATINE INSENSITIVE 1 (COI1). AtMYB44 over-expression down-regulated defense responses against the necrotrophic pathogen Alternaria brassicicola, but up-regulated WRKY70 and PR genes, leading to enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000. The knockout mutant atmyb44 shows opposite effects. Induction of WRKY70 by SA is reduced in atmyb44 and npr1-1 mutants, and is totally abolished in atmyb44 npr1-1 double mutants, showing that WRKY70 is regulated independently through both NPR1 and AtMYB44. AtMYB44 over-expression does not change SA content, but AtMYB44 over-expression phenotypes, such as retarded growth, up-regulated PR1 and down-regulated PDF1.2 are reversed by SA depletion. The wrky70 mutation suppressed AtMYB44 over-expression phenotypes, including up-regulation of PR1 expression and down-regulation of PDF1.2 expression. β-estradiol-induced expression of AtMYB44 led to WRKY70 activation and thus PR1 activation. AtMYB44 binds to the WRKY70 promoter region, indicating that AtMYB44 acts as a transcriptional activator of WRKY70 by directly binding to a conserved sequence element in the WRKY70 promoter. These results demonstrate that AtMYB44 modulates antagonistic interaction by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  15. Cyclooxygenase-2 induction in macrophages is modulated by docosahexaenoic acid via interactions with free fatty acid receptor 4 (FFA4).

    Science.gov (United States)

    Li, Xinzhi; Yu, Ying; Funk, Colin D

    2013-12-01

    Cyclooxygenase-2 (COX-2)-derived prostaglandins are implicated in numerous inflammatory disorders. The purpose of these studies was to examine previously unexplored interactions between COX-2 induction and docosahexaenoic acid (DHA) via the free fatty acid receptor 4 (FFA4) signaling pathway in murine RAW 264.7 cells and peritoneal macrophages challenged with lipopolysaccharide (LPS). DHA dose (IC50=18 μM)- and time-dependently reduced COX-2 expression, without affecting COX-1. DHA (25 μM for 24 h) decreased LPS-induced prostaglandin E2 (PGE2) synthesis by 81%, primarily through reducing COX-2 (60%), as well as down-regulating microsomal prostaglandin E synthase-1 (46%), but independently of peroxisome proliferator-activated receptors. FFA4 knockdown abrogated DHA effects on COX-2 induction, PGE2 production, and interleukin 6 (IL-6) gene expression. In the presence of inhibitors of eicosanoid metabolism via COX-2, 12/15-lipoxygenase and CYP450s (rofecoxib (1 μM), PD146176 (2 μM), or MS-PPOH (20 μM)), DHA was still effective in attenuating COX-2 induction. Moreover, Toll-like receptor 4 signaling via Akt/JNK phosphorylation and p65 nuclear translocation was repressed by DHA-activated FFA4 coupling with β-arrestin 2, which was reversed by FFA4 knockdown. These data support DHA modulation of COX-2 expression and activity, in part, via FFA4, which provides a new mechanistic explanation for some of the anti-inflammatory effects of DHA.

  16. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  17. Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci.

    Science.gov (United States)

    Cheng, Xingqun; Redanz, Sylvio; Cullin, Nyssa; Zhou, Xuedong; Xu, Xin; Joshi, Vrushali; Koley, Dipankar; Merritt, Justin; Kreth, Jens

    2018-01-15

    Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H 2 O 2 , which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H 2 O 2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H 2 O 2 production by S. sanguinis and S. gordonii S. sanguinis H 2 O 2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H 2 O 2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H 2 O 2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H 2 O 2 IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H 2 O 2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival

  18. Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes

    DEFF Research Database (Denmark)

    Rodriguez De Turco, Elena B; Jackson, Fannie R; DeCoster, Mark A

    2002-01-01

    The lipid mediators generated by phospholipases A(2) (PLA(2)), free arachidonic acid (AA), eicosanoids, and platelet-activating factor, modulate neuronal activity; when overproduced, some of them become potent neurotoxins. We have shown, using primary cortical neuron cultures, that glutamate...... and secretory PLA(2) (sPLA(2)) from bee venom (bv sPLA(2)) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA(2) are selective ligands of cell-surface sPLA(2) receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate......) and in minor changes in other phospholipids. A similar profile, although of greater magnitude, was observed 20 hr posttreatment. Glutamate (80 microM) induced much less mobilization of (3)H-AA than did sPLA(2) and resulted in a threefold greater degradation of (3)H-AA PE than of (3)H-AA PC by 20 hr...

  19. Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels.

    Science.gov (United States)

    Herman, Mark A; She, Pengxiang; Peroni, Odile D; Lynch, Christopher J; Kahn, Barbara B

    2010-04-09

    Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

  20. Atrazine biodegradation modulated by clays and clay/humic acid complexes

    International Nuclear Information System (INIS)

    Besse-Hoggan, Pascale; Alekseeva, Tatiana; Sancelme, Martine; Delort, Anne-Marie; Forano, Claude

    2009-01-01

    The fate of pesticides in the environment is strongly related to the soil sorption processes that control not only their transfer but also their bioavailability. Cationic (Ca-bentonite) and anionic (Layered Double Hydroxide) clays behave towards the ionisable pesticide atrazine (AT) sorption with opposite tendencies: a noticeable sorption capacity for the first whereas the highly hydrophilic LDH showed no interactions with AT. These clays were modified with different humic acid (HA) contents. HA sorbed on the clay surface and increased AT interactions. The sorption effect on AT biodegradation and on its metabolite formation was studied with Pseudomonas sp. ADP. The biodegradation rate was greatly modulated by the material's sorption capacity and was clearly limited by the desorption rate. More surprisingly, it increased dramatically with LDH. Adsorption of bacterial cells on clay particles facilitates the degradation of non-sorbed chemical, and should be considered for predicting pesticide fate in the environment. - The biodegradation rate of atrazine was greatly modulated by adsorption of the pesticide and also bacterial cells on clay particles.

  1. Tannic Acid as a Potential Modulator of Norfloxacin Resistance in Staphylococcus Aureus Overexpressing norA.

    Science.gov (United States)

    Diniz-Silva, Helena Taina; Cirino, Isis Caroline da Silva; Falcão-Silva, Vivyanne Dos Santos; Magnani, Marciane; de Souza, Evandro Leite; Siqueira-Júnior, José P

    2016-01-01

    Tannins have shown inhibitory effects against pathogenic bacteria, and these properties make tannins potential modifying agents in bacterial resistance. The minimum inhibitory concentration (MIC) of tannic acid (TA), gallic acid (GA) and norfloxacin (Nor) against Staphylococcus aureus SA-1119 (NorA-effluxing strain) was determined using broth microdilution tests. To assess the modulation of antibiotic resistance, the MIC of Nor was determined in growth media with or without TA or GA at a subinhibitory concentration (1/4 MIC). The checkerboard method was performed to obtain the fractional inhibitory concentration index (FICI) for the combined application of TA and Nor. TA displayed a weak inhibitory effect (MIC 512 μg/ml) against S. aureus SA-1119, while no inhibitory effect was displayed by GA (MIC >512 μg/ml). However, when TA was tested at a subinhibitory concentration in combination with Nor, the MIC of Nor against S. aureus SA-1119 decreased from 128 to 4 μg/ml (32-fold); this effect was not observed for GA. In the checkerboard assay, the MIC of TA and Nor decreased from 512 to 128 μg/ml (4-fold) and from 128 to 8 μg/ml (16-fold), respectively. The combination of TA and Nor presented an FICI as low as 0.31, which indicates a synergistic interaction. TA is a potential agent for increasing the clinical efficacy of Nor to control resistant S. aureus. © 2016 S. Karger AG, Basel.

  2. Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    2007-02-01

    Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

  3. Alkaline protease production by alkaliphilic marine bacteria isolated ...

    African Journals Online (AJOL)

    The molecular mass determined using SDS-PAGE, was nearly 31.0 39 kDa. Some fundamental properties like effects of different temperatures, pH, metal ions (Ca2+, Mg2+, Cu2+, Pb3+, Mn2+ and Cd2+) and ethylene diamine tetraacetic acid (EDTA) on protease activity were also studied. Maximum activities were obtained ...

  4. Effects of Protease, Phytase and a Bacillus sp. Direct-Fed Microbial on Nutrient and Energy Digestibility, Ileal Brush Border Digestive Enzyme Activity and Cecal Short-Chain Fatty Acid Concentration in Broiler Chickens

    Science.gov (United States)

    Murugesan, Ganapathi R.; Romero, Luis F.; Persia, Michael E.

    2014-01-01

    Two experiments were conducted to determine the effects of protease and phytase (PP) and a Bacillus sp. direct-fed microbial (DFM) on dietary energy and nutrient utilization in broiler chickens. In the first experiment, Ross 308 broiler chicks were fed diets supplemented with PP and DFM in a 2×2 factorial arrangement. The 4 diets (control (CON), CON + PP, CON + DFM, and CON + PP + DFM) were fed from 15–21 days of age. In Experiment 1, significant interaction (P≤0.01) between PP and DFM on the apparent ileal digestibility coefficient for starch, crude protein, and amino acid indicated that both additives increased the digestibility. Both additives increased the nitrogen retention coefficient with a significant interaction (P≤0.01). Although no interaction was observed, significant main effects (P≤0.01) for nitrogen-corrected apparent ME (AMEn) for PP or DFM indicated an additive response. In a follow-up experiment, Ross 308 broiler chicks were fed the same experimental diets from 1–21 days of age. Activities of ileal brush border maltase, sucrase, and L-alanine aminopeptidase were increased (P≤0.01) by PP addition, while a trend (P = 0.07) for increased sucrase activity was observed in chickens fed DFM, in Experiment 2. The proportion of cecal butyrate was increased (P≤0.01) by DFM addition. Increased nutrient utilization and nitrogen retention appear to involve separate but complementary mechanisms for PP and DFM, however AMEn responses appear to have separate and additive mechanisms. PMID:25013936

  5. Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Drew, BG; Carey, AL; Natoli, AK

    2011-01-01

    We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study inve...

  6. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

  7. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  8. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

    2010-07-15

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  9. Modulation of the acidity of niobic acid by ion-doping: Effects of nature and amount of the dopant ions

    Energy Technology Data Exchange (ETDEWEB)

    Carniti, Paolo [Dipartimento di Chimica, Università degli Studi di Milano (UNIMI), Milano (Italy); Gervasini, Antonella, E-mail: antonella.gervasini@unimi.it [Dipartimento di Chimica, Università degli Studi di Milano (UNIMI), Milano (Italy); Marzo, Matteo [Dipartimento di Chimica, Università degli Studi di Milano (UNIMI), Milano (Italy); Auroux, Aline [Université Lyon 1, CNRS, UMR 5256, (IRCELYON), Villeurbanne (France)

    2013-09-10

    Highlights: ► Mitigation of the surface acidity of niobic acid was pursued by K-, Ba-, and Nd-doping. ► Thermal techniques of study were effective for the acidity study. ► The nature of the dopant influences the effectiveness of the acidity tuning of niobic acid. ► The acidity of the doped surfaces decreased with increasing the dopant species added to niobic acid. ► The samples showed different acidity when measured in gas–solid phase (intrinsic acidity) and water (effective acidity). - Abstract: The acidity of niobic acid (NBO) has been successfully mitigated and tuned by addition of K{sup +}, Ba{sup 2+} and Nd{sup 3+} dopant species in amounts from 1 to 15 atom nm{sup −2}. The characterization of the intrinsic acid properties of the samples was performed by adsorption of NH{sub 3} in a volumetric–microcalorimetric coupled line and by temperature programmed desorption (TPD) of 2-phenylethylamine in a thermogravimetric apparatus. The K-dopant was more effective in decreasing the acidity of niobic acid than the Ba- and Nd-dopants. Complementary measurements of the effective acidity of the samples in water by base titrations with 2-phenylethylamine completed the study and revealed a different picture of the effect of the three dopants on the NBO acidity in water. All the results indicated that the K-dopant targeted more selectively the Brønsted acid sites, acting as an ion-exchanger, while Ba- and Nd-species predominantly acted on the Lewis acid sites of the NBO surface.

  10. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    Science.gov (United States)

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

    Science.gov (United States)

    Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

    2010-11-18

    The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

  12. Structure of the Integral Membrane Protein CAAX Protease Ste24p

    Energy Technology Data Exchange (ETDEWEB)

    Pryor Jr., Edward E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Horanyi, Peter S. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Clark, Kathleen M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Fedoriw, Nadia [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Connelly, Sara M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Koszelak-Rosenblum, Mary [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Zhu, Guangyu [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Malkowski, Michael G. [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); State Univ. of New York, Buffalo, NY (United States); Wiener, Michael C. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Dumont, Mark E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States)

    2012-10-26

    Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

  13. Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases.

    Science.gov (United States)

    Luo, Jie; Xiao, Chen; Zhang, Hao; Ren, Fazheng; Lei, Xingen; Yang, Zibiao; Yu, Zhengquan

    2018-04-01

    The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Salicylic Acid Alleviates Aluminum Toxicity in Soybean Roots through Modulation of Reactive Oxygen Species Metabolism

    Directory of Open Access Journals (Sweden)

    Ning Liu

    2017-11-01

    Full Text Available As an important signal molecule, salicylic acid (SA improves plant tolerance to aluminum (Al stress. The objective of this study was to investigate the effects of exogenous SA application on the dynamics of endogenous SA and reactive oxygen species in soybean (Glycine max L. exposed to Al stress. The roots of soybean seedlings were exposed to a combination of AlCl3 (30 μM and SA (10 μM/PAC (100 μM, paclobutrazol, SA biosynthesis inhibitor for 3, 6, 9, and 12 h. Al stress induced an increase in endogenous SA concentration in a time-dependent manner, also verified by the up-regulated expression of GmNPR1, an SA-responsive gene. Al stress increased the activities of phenylalanine ammonia-lyase (PAL and benzoic acid 2-hydroxylase (BA2H, and the contents of SA, O2- and malondialdehyde (MDA in the root apex. The application of exogenous SA increased PAL and BA2H, and reduced O2- and MDA contents in soybean roots under Al stress. PAC inhibited the SA induced increase in BA2H activity. In addition, the SA application resulted in a rapid increase in hydrogen peroxide (H2O2 concentration under Al stress, followed by a sharp decrease. Compared with the plants exposed to Al alone, Al+SA plants possessed higher activities of superoxide dismutase, peroxidase, and ascorbate peroxidase, and lower catalase activity, indicating that SA alleviated Al-induced oxidative damage. These results suggested that PAL and BA2H were involved in Al-induced SA production and showed that SA alleviated the adverse effects of Al toxicity by modulating the cellular H2O2 level and the antioxidant enzyme activities in the soybean root apex.

  15. Salicylic acid alleviates aluminum toxicity in soybean roots through modulation of reactive oxygen species metabolism

    Science.gov (United States)

    Liu, Ning; Song, Fengbin; Zhu, Xiancan; You, Jiangfeng; Yang, Zhenming; Li, Xiangnan

    2017-11-01

    As an important signal molecule, salicylic acid (SA) improves plant tolerance to aluminum (Al) stress. The objective of this study was to investigate the effects of exogenous SA application on the dynamics of endogenous SA and reactive oxygen species in soybean (Glycine max L.) exposed to Al stress. The roots of soybean seedlings were exposed to a combination of AlCl3 (30 μM) and SA (10 μM)/PAC (100 μM, paclobutrazol, SA biosynthesis inhibitor) for 3, 6, 9 and 12 h. Al stress induced an increase in endogenous SA concentration in a time-dependent manner, also verified by the up-regulated expression of GmNPR1, an SA-responsive gene. Al stress increased the activities of phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H), and the contents of SA, O2- and malondialdehyde (MDA) in the root apex. The application of exogenous SA increased PAL and BA2H, and reduced O2- and MDA contents in soybean roots under Al stress. PAC inhibited the SA induced increase in BA2H activity. In addition, the SA application resulted in a rapid increase in hydrogen peroxide (H2O2) concentration under Al stress, followed by a sharp decrease. Compared with the plants exposed to Al alone, Al+SA plants possessed higher activities of superoxide dismutase, peroxidase and ascorbate peroxidase, and lower catalase activity, indicating that SA alleviated Al-induced oxidative damage. These results suggested that PAL and BA2H were involved in Al-induced SA production and showed that SA alleviated the adverse effects of Al toxicity by modulating the cellular H2O2 level and the antioxidant enzyme activities in the soybean root apex.

  16. [ACID-BASE MODULATION OF LYSOZYME ACTIVITY IN MEDIUM FOR CULTIVATION OF ENTEROBACTERIA].

    Science.gov (United States)

    Andryuschenko, S V; Perunova, N B

    2015-01-01

    Determination of modulating effect of acid-base state of medium for cultivation of enterobacteria on activity of C-type lysozyme. Escherichia coli BL21 (DE3) strain for protein expression, Escherichia coli K12 MG1655 model strain, Escherichia coli No. 242 strain, isolated from intestine biotope; 2 Klebsiella pneumoniae strains, one of those contained plasmid homologue of periplasmatic lysozyme inhibitor gene pliC; 1 typical Salmonella enterica ATCC 14028 strain and a Micrococcus luteus ATCC 15307 strain as a control--served as material for the study. The bacteria were cultivated for 24 hours in 2 ml of liquid medium LB at 37 degrees C, 250 rpm. Determination of antilysozyme activity (ALA) was carried out by a photonepehlometrical method according to O.V. Bukharin et al. (1999) with alterations. All the studied microorganisms, including Micrococcus luteus, at the specified conditions 24 hours after cultivation were established to change the pH of the liquid nutrient medium LB from the initial value of 6.6 ± 0.1 to 8.2 ± 0.2 units. ALA determination in the cultivation medium without buffer correction was accompanied by a decline of lysozyme activity at an order of magnitude. The effect was absent during ALA measurement by a standard technique. The local shift of acid-base state of biotope under the conditions of buffer system insufficiency results in a reversible alteration of antimicrobial activity of muramidase, that among other non-specific factors of the environment determines the background of interactions on the level of associative symbiosis. This aspect should be taken into consideration during development of models, that are close to real conditions of microsymbiocenotical interactions.

  17. Protease activation involved in resistance of human cells to x-ray cell killing

    International Nuclear Information System (INIS)

    Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo

    2003-01-01

    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

  18. Role of abscisic acid (aba) in modulating the responses of two apple rootstocks to drought stress

    International Nuclear Information System (INIS)

    Zhang, L.; Li, X.; Li, B.; Han, M.; Liu, F.; Zhang, L.; Zheng, P.

    2014-01-01

    Drought stress is considered as the main limiting factor for apple (Malus domestica L.) production in some semi-arid areas of China. In this study, we investigated the modulation role of abscisic acid (ABA) and fluridone (ABA synthesis inhibitor) on water relations and antioxidant enzyme system in 2-year-old seedlings of two apple rootstocks i.e. Malus sieversii (Ledeb.) Roem. (MS) and Malus hupehensis (Pamp.) Rehd. (MH). Drought stress induced ion leakage, accumulation of malondiadehyde (MDA) and decreases in leaf water potential and relative water content (RWC) in both rootstocks, which were significantly alleviated by exogenous ABA application. Drought stress also induced markedly increases in endogenous ABA content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR), to a greater magnitude in MS as compared to MH rootstock. Concentration of 100 mol/L and 50 mol/L ABA had the most positive effects on drought-stressed rootstocks of MS and MH, respectively. Spraying optimum exogenous ABA contributed to enhancement in most of the above antioxidant enzymes activities but reduction in content of MDA and maintained the appropriate leaf water potential and RWC in both rootstocks. Pretreatment with fluridone aggravated ion leakage and the accumulation of MDA in two apple rootstocks under drought stress, which was overcome by exogenous ABA application to some extent. In conclusion, the endogenous ABA was probably involved in the regulation of two apple rootstocks in responses to drought stress. (author)

  19. Modulation of the nitrate reductase transcript by cytokinin and abscisic acid in etiolated barley seedlings

    International Nuclear Information System (INIS)

    Lu, Jia-ling; Enl, J.R.; Chen, Chong-maw

    1989-01-01

    To investigate the molecular mechanism of the hormonal modulation of nitrate reductase (NR) activity, the influence of benzyladenine (BA) and/or abscisic acid (ABA) on the level of NR poly(A)RNA was studied in etiolated barley seedlings using a 32 P-labelled NR cDNA as a probe. Enhancement of NR activity by 2 x 10 -5 M BA was measurable only after 60 minutes of exposure of the seedlings to light, while a significant stimulatory effect on the transcript level could by clearly detected within 15 minutes. Northern blot analyses of the levels of NR poly(A)RNA indicate that the amount present is proportional to the concentration of BA applied to the seedlings. The stimulatory effects seen for BA were nullified by ABA. The counteractive effects of ABA on BA were dose-responsive, with greater inhibition at higher concentrations of ABA. Evidence suggests that the interaction of BA and ABA on NR activity is at the transcriptional level, however, is also possible that interactions occur at the postranscriptional level as well

  20. Modulation of benzodiazepine by lysine and pipecolic acid on pentylenetetrazol-induced seizures

    International Nuclear Information System (INIS)

    Chang, Y.F.; Hargest, V.; Chen, J.S.

    1988-01-01

    L-lysine and its metabolite pipecolic acid (PA) have been studied for their effects on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine of L-Pa i.p. significantly increased clonic and tonic latencies in a dose-dependent manner against 90 mg/kg PTZ-induced seizures. L-Lysine but not L-Pa enhanced the anticonvulsant effect of diazepam (DZ). L-Pa i.c.v. showed a slight decrease in clonic latency; it did not enhance the antiseizure activity of DZ; it caused seizures at 0.6 mmol/kg. D-PA i.c.v. displayed an opposite effect compared to its L-isomer. The anticonvulsant effect of L-lysine in terms of increase in seizure latency and survival was even more amplified when tested with a submaximal PTZ concentration. L-Lysine showed an enhancement of specific 3 H-flunitrazepam(FZ) binding to mouse brain membranes both in vitro an din vivo. The possibility of L-lysine acting as a modulator for the GABA/benzodiazepine receptors was demonstrated. Since L-PA showed enhancement of 3 H-FZ binding only in vitro but not in vivo, the anticonvulsant effect of L-PA may not be linked to the GABA/benzodiazepine receptor

  1. Modulation of Tumor Cell Metabolism by Laser Photochemotherapy with Cisplatin or Zoledronic Acid In Vitro.

    Science.gov (United States)

    Heymann, Paul Günther Baptist; Henkenius, Katharina Sabine Elisabeth; Ziebart, Thomas; Braun, Andreas; Hirthammer, Klara; Halling, Frank; Neff, Andreas; Mandic, Robert

    2018-03-01

    Laser photochemotherapy is a new approach in cancer treatment using low-level laser therapy (LLLT) to enhance the effect of chemotherapy. In order to evaluate the effect of LLLT on tumor cells, HeLa cells were treated with cisplatin or zoledronic acid (ZA) followed by LLLT. Cell viability was evaluated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Oxidative phosphorylation and glycolysis were measured using extracellular flux analysis. Immunocytochemistry of heat-shock protein 70 (HSP70) and western blot analysis were performed. LLLT alone increased viability and was associated with lower oxidative phosphorylation but higher glycolysis rates. Cisplatin and ZA alone lowered cell viability, glycolysis and oxidative phosphorylation. This effect was significantly enhanced in conjunction with LLLT and was accompanied by reduced oxidative phosphorylation and collapse of glycolysis. Our observations indicate that LLLT may raise the cytotoxicity of cisplatin and ZA by modulating cellular metabolism, pointing to a possible application in cancer treatment. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. Bile Acid Malabsorption After Pelvic and Prostate Intensity Modulated Radiation Therapy: An Uncommon but Treatable Condition

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Victoria [Academic Urology Unit, Institute of Cancer Research and The Royal Marsden Hospital, London and Sutton (United Kingdom); Benton, Barbara [Gastroenterology Unit, Institute of Cancer Research and The Royal Marsden Hospital, London and Sutton (United Kingdom); Sohaib, Aslam [Department of Radiology, Institute of Cancer Research and The Royal Marsden Hospital, London and Sutton (United Kingdom); Dearnaley, David [Academic Urology Unit, Institute of Cancer Research and The Royal Marsden Hospital, London and Sutton (United Kingdom); Andreyev, H. Jervoise N., E-mail: j@andreyev.demon.co.uk [Gastroenterology Unit, Institute of Cancer Research and The Royal Marsden Hospital, London and Sutton (United Kingdom)

    2012-12-01

    Purpose: Intensity modulated radiation therapy (IMRT) is a significant therapeutic advance in prostate cancer, allowing increased tumor dose delivery and increased sparing of normal tissues. IMRT planning uses strict dose constraints to nearby organs to limit toxicity. Bile acid malabsorption (BAM) is a treatable disorder of the terminal ileum (TI) that presents with symptoms similar to radiation therapy toxicity. It has not been described in patients receiving RT for prostate cancer in the contemporary era. We describe new-onset BAM in men after IMRT for prostate cancer. Methods and Materials: Diagnosis of new-onset BAM was established after typical symptoms developed, selenium-75 homocholic acid taurine (SeHCAT) scanning showed 7-day retention of <15%, and patients' symptoms unequivocally responded to a bile acid sequestrant. The TI was identified on the original radiation therapy plan, and the radiation dose delivered was calculated and compared with accepted dose-volume constraints. Results: Five of 423 men treated in a prospective series of high-dose prostate and pelvic IMRT were identified with new onset BAM (median age, 65 years old). All reported having normal bowel habits before RT. The volume of TI ranged from 26-141 cc. The radiation dose received by the TI varied between 11.4 Gy and 62.1 Gy (uncorrected). Three of 5 patients had TI treated in excess of 45 Gy (equivalent dose calculated in 2-Gy fractions, using an {alpha}/{beta} ratio of 3) with volumes ranging from 1.6 cc-49.0 cc. One patient had mild BAM (SeHCAT retention, 10%-15%), 2 had moderate BAM (SeHCAT retention, 5%-10%), and 2 had severe BAM (SeHCAT retention, <5%). The 3 patients whose TI received {>=}45 Gy developed moderate to severe BAM, whereas those whose TI received <45 Gy had only mild to moderate BAM. Conclusions: Radiation delivered to the TI during IMRT may cause BAM. Identification of the TI from unenhanced RT planning computed tomography scans is difficult and may impede

  3. Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

    Science.gov (United States)

    Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E

    2014-06-01

    The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

  4. The action of neutrophil serine proteases on elastin and its precursor

    DEFF Research Database (Denmark)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther

    2012-01-01

    This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

  5. Modulation of salt (NaCl)-induced effects on oil composition and fatty acid profile of sunflower (Helianthus annuus L.) by exogenous application of salicylic acid.

    Science.gov (United States)

    Noreen, Sibgha; Ashraf, Muhammad

    2010-12-01

    Salicylic acid (SA) is a potential endogenous plant hormone that plays an important role in plant growth and development. Since sunflower yield and its seed oil yield are adversely affected by salinity, in this study the role of SA in modulating salt (NaCl)-induced effects on various yield and oil characteristics of sunflower was investigated. For this purpose a greenhouse experiment comprising two sunflower hybrid lines (Hysun-33 and SF-187), two NaCl levels (0 and 120 mmol L(-1)) and four SA levels (0, 100, 200 and 300 mg L(-1)) was conducted. Salt stress markedly reduced yield, oil content, linoleic acid and δ-tocopherol in both sunflower lines, while it increased linolenic acid, palmitic acid, stearic acid and α- and γ-tocopherols. However, increasing levels of foliar-applied SA resulted in improved achene yield and hundred-achene weight in both lines. Foliar-applied SA caused a significant decrease in oil stearic acid and α- and γ-tocopherols in both lines under non-saline and saline conditions. Salt-induced harmful effects on achene yield and oil characteristics of sunflower could be alleviated by exogenous application of SA. High doses of SA caused a marked increase in sunflower achene oil content as well as some key fatty acids. Copyright © 2010 Society of Chemical Industry.

  6. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

    PURPOSE OF REVIEW: To review the evidence linking use of HIV protease inhibitors with excess risk of cardiovascular disease (CVD) in HIV+ populations. RECENT FINDINGS: For the two contemporary most frequently used protease inhibitors, darunavir and atazanavir [both pharmacologically boosted...

  7. Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

    Science.gov (United States)

    Song, Qin-Qin; Lu, Ming-Zhi; Song, Juan; Chi, Miao-Miao; Sheng, Lin-Jun; Yu, Jie; Luo, Xiao-Nuan; Zhang, Lu; Yao, Hai-Lan; Han, Jun

    2015-10-02

    To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4

    DEFF Research Database (Denmark)

    Watterson, Kenneth R; Hansen, Steffen V F; Hudson, Brian D

    2017-01-01

    High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential...... of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many...

  9. Modulation of Antioxidant Enzymatic Activities by Certain Antiepileptic Drugs (Valproic Acid, Oxcarbazepine, and Topiramate): Evidence in Humans and Experimental Models

    Science.gov (United States)

    Cárdenas-Rodríguez, Noemí; Coballase-Urrutia, Elvia; Rivera-Espinosa, Liliana; Romero-Toledo, Arantxa; Sampieri, Aristides III; Ortega-Cuellar, Daniel; Montesinos-Correa, Hortencia; Floriano-Sánchez, Esaú; Carmona-Aparicio, Liliana

    2013-01-01

    It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA) receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity). This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS) activation and the generation of reactive oxygen species (ROS). Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs) exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA), oxcarbazepine (OXC), and topiramate (TPM) modulate oxidative stress. PMID:24454986

  10. Modulation of Antioxidant Enzymatic Activities by Certain Antiepileptic Drugs (Valproic Acid, Oxcarbazepine, and Topiramate: Evidence in Humans and Experimental Models

    Directory of Open Access Journals (Sweden)

    Noemí Cárdenas-Rodríguez

    2013-01-01

    Full Text Available It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity. This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS activation and the generation of reactive oxygen species (ROS. Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA, oxcarbazepine (OXC, and topiramate (TPM modulate oxidative stress.

  11. Validity and Practitality of Acid-Base Module Based on Guided Discovery Learning for Senior High School

    Science.gov (United States)

    Yerimadesi; Bayharti; Jannah, S. M.; Lufri; Festiyed; Kiram, Y.

    2018-04-01

    This Research and Development(R&D) aims to produce guided discovery learning based module on topic of acid-base and determine its validity and practicality in learning. Module development used Four D (4-D) model (define, design, develop and disseminate).This research was performed until development stage. Research’s instruments were validity and practicality questionnaires. Module was validated by five experts (three chemistry lecturers of Universitas Negeri Padang and two chemistry teachers of SMAN 9 Padang). Practicality test was done by two chemistry teachers and 30 students of SMAN 9 Padang. Kappa Cohen’s was used to analyze validity and practicality. The average moment kappa was 0.86 for validity and those for practicality were 0.85 by teachers and 0.76 by students revealing high category. It can be concluded that validity and practicality was proven for high school chemistry learning.

  12. Distinctive Roles of D-Amino Acids in the Homochiral World: Chirality of Amino Acids Modulates Mammalian Physiology and Pathology.

    Science.gov (United States)

    Sasabe, Jumpei; Suzuki, Masataka

    2018-05-22

    Living organisms enantioselectively employ L-amino acids as the molecular architecture of protein synthesized in the ribosome. Although L-amino acids are dominantly utilized in most biological processes, accumulating evidence points to the distinctive roles of D-amino acids in non-ribosomal physiology. Among the three domains of life, bacteria have the greatest capacity to produce a wide variety of D-amino acids. In contrast, archaea and eukaryotes are thought generally to synthesize only two kinds of D-amino acids: D-serine and D-aspartate. In mammals, D-serine is critical for neurotransmission as an endogenous coagonist of N-methyl D-aspartate receptors. Additionally, D-aspartate is associated with neurogenesis and endocrine systems. Furthermore, recognition of D-amino acids originating in bacteria is linked to systemic and mucosal innate immunity. Among the roles played by D-amino acids in human pathology, the dysfunction of neurotransmission mediated by D-serine is implicated in psychiatric and neurological disorders. Non-enzymatic conversion of L-aspartate or L-serine residues to their D-configurations is involved in age-associated protein degeneration. Moreover, the measurement of plasma or urinary D-/L-serine or D-/L-aspartate levels may have diagnostic or prognostic value in the treatment of kidney diseases. This review aims to summarize current understanding of D-amino-acid-associated biology with a major focus on mammalian physiology and pathology.

  13. Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice

    OpenAIRE

    Chaplin, Alice; Parra, Pilar; Serra, Francisca; Palou, Andreu

    2015-01-01

    The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute...

  14. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

    Budak, Sebnem Ozturkoglu; Zhou, M.; Brouwer, Carlo; Wiebenga, A.; Benoit, Isabelle; Di Falco, Marcos; Tsang, Adrian; de Vries, Ronald P; van den Brink, J.

    2014-01-01

    BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide

  15. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  16. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

  17. Influencia de especies forestales sobre la actividad de las enzimas fosfatasa ácida y proteasas en un suelo de bosque Influence of tree species on the activity of acid phosphatase and protease in a forest soil

    Directory of Open Access Journals (Sweden)

    Rl Defrieri

    2008-12-01

    reflejaron mejor los cambios debidos a la influencia de las diferentes especies y de la época del año que otros parámetros químicos del suelo.Plant cover and especially the dominant tree species affect biological and chemical properties of the soil. Litter decomposition rate is affected by its N and P concentration. The aim of this work was to determine the different effects of forest tree species on some biochemical properties of the soil. The study site was located at the Reserva Natural Estricta Colonia Benítez, Chaco, Argentina. Soil samples were taken under trees of the four dominant species in the area and at two depths (0-10 cm and 10-20cm and moments: in summer and in winter. Activities of acid phosphatase and protease enzymes and some edaphic parameters were determined. The results obtained for all studied variables were significantly lower at the 10-20 cm depth, for all forest species and in both seasons. Values of enzyme activities showed significant differences between species only in surface samples where the incorporation of organic matter is greater and in summer. In these conditions, the values of enzymatic activities obtained in soils under each species ranged between 1,600 and 900 μg p-nitrophenol g-1 h-1 for acid phosphatase and between 850 y 450 g tyrosine g-1h-1 for protease. For two of the studied species, a relationship was found between the amount of litter produced, the different decomposition rates and the N and P concentrations in senescent leaves with the enzyme activities in soils. Inorganic N and available P concentrations in soils did not show significant differences between species. In this study, soil enzyme activities were more related to the overlying species than some measured soil parameters.

  18. Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Hongxia Cui

    2015-01-01

    Full Text Available A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China, was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0 and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF but mildly increased (~107% in the presence of ethylenediaminetetraacetic acid (EDTA, indicating that the production contains serine-protease(s and nonmetal protease(s. Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+ that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS, an oxidizing agent (1% H2O2, and several organic solvents (methanol, isopropanol, and acetone. These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

  19. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  20. Factor VII-activating protease

    DEFF Research Database (Denmark)

    Ramanathan, Ramshanker; Gram, Jørgen B; Sand, Niels Peter R

    2017-01-01

    : Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens...

  1. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

    A trial was conducted to evaluate whether the addition of commercial enzyme preparations containing carbohydrases and a protease would increase the available metabolizable energy (ME) of maize-soya-based broiler diets. Seven thousand five hundred and sixty (7560) day-old Ross 788 chicks were randomly allocated ...

  2. Modulation of hepatic biotransformation and biliary excretion of bile acid by age and sinusoidal bile acid load

    International Nuclear Information System (INIS)

    Baumgartner, U.; Miyai, K.; Hardison, W.G.M.

    1987-01-01

    Pericentral hepatocytes excrete bile acids more slowly and biotransform them more than periportal cells. This may reflect adaptation to low pericentral bile acid concentration or may be intrinsic. The authors studied two models in which pericentral bile acid concentrations are high: the 72-h choledocho-caval shunt (CCS) rat and the 3- to 4-wk-old rat. Livers were perfused forward or backward to assess periportal or pericentral hepatocyte function. Taurodeoxycholate (TDC) was infused at 32 nmol x min -1 x g liver -1 , and a bolus of [ 3 H]TDC was given to assess metabolism and excretion of bile acids. In CCS livers perfused backward, pericentral cells resembled periportal cells of controls in that time to excrete 50% of administered [ 3 H]-TDC (t 50 ) was reduced by two-thirds and [ 3 H]TDC biotransformation was reduced by about half. In young livers t 50 was half that of adult livers when perfused backward. Biotransformation, however, was not reduced. Young livers biotransformed more than adult controls for any given residence time of bile acid in the liver. They conclude that the difference between pericentral and perioportal cells as regards bile acid processing is adaptive. Livers from young rats biotransform more bile acid than those from controls under similar conditions

  3. Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.

    Science.gov (United States)

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-09-02

    An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Effect of Semen on Vaginal Fluid Cytokines and Secretory Leukocyte Protease Inhibitor

    Directory of Open Access Journals (Sweden)

    Kathy J. Agnew

    2008-01-01

    Methods: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test. Results: of 138 subjects, 28 (20% had acid phosphatase detected; of these, only 19 (68% reported recent intercourse and 3 (11% had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase. Conclusions: proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal SLPI concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations.

  5. Gallic acid modulates phenotypic behavior and gene expression in oral squamous cell carcinoma cells by interfering with leptin pathway.

    Science.gov (United States)

    Santos, Eliane Macedo Sobrinho; da Rocha, Rogério Gonçalves; Santos, Hércules Otacílio; Guimarães, Talita Antunes; de Carvalho Fraga, Carlos Alberto; da Silveira, Luiz Henrique; Batista, Paulo Ricardo; de Oliveira, Paulo Sérgio Lopes; Melo, Geraldo Aclécio; Santos, Sérgio Henrique; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena; Farias, Lucyana Conceição

    2018-01-01

    Gallic acid is a polyphenolic compost appointed to interfere with neoplastic cells behavior. Evidence suggests an important role of leptin in carcinogenesis pathways, inducing a proliferative phenotype. We investigated the potential of gallic acid to modulate leptin-induced cell proliferation and migration of oral squamous cell carcinoma cell lines. The gallic acid effect on leptin secretion by oral squamous cell carcinoma cells, as well as the underlying molecular mechanisms, was also assessed. For this, we performed proliferation, migration, immunocytochemical and qPCR assays. The expression levels of cell migration-related genes (MMP2, MMP9, Col1A1, and E-cadherin), angiogenesis (HIF-1α, mir210), leptin signaling (LepR, p44/42 MAPK), apoptosis (casp-3), and secreted leptin levels by oral squamous cell carcinoma cells were also measured. Gallic acid decreased proliferation and migration of leptin-treated oral squamous cell carcinoma cells, and reduced mRNA expression of MMP2, MMP9, Col1A1, mir210, but did not change HIF-1α. Gallic acid decreased levels of leptin secreted by oral squamous cell carcinoma cells, accordingly with downregulation of p44/42 MAPK expression. Thus, gallic acid appears to break down neoplastic phenotype of oral squamous cell carcinoma cells by interfering with leptin pathway. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    Directory of Open Access Journals (Sweden)

    Jiangning Song

    Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

  7. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human ca...

  8. Differential saturation study of radial and angular modulation mechanisms of electron spin--lattice relaxation for trapped hydrogen atoms in sulfuric acid glasses. [X radiation

    Energy Technology Data Exchange (ETDEWEB)

    Plonka, A; Kevan, L

    1976-11-01

    A differential ESR saturation study of allowed transitions and forbidden proton spin-flip satellite transitions for trapped hydrogen atoms in sulfuric acid glasses indicates that angular modulation dominates the spin-lattice relaxation mechanisms and suggests that the modulation arises from motion of the H atom.

  9. Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus

    Directory of Open Access Journals (Sweden)

    Emyr Peña

    2015-09-01

    Full Text Available Partial characterizations of digestive proteases were studied in three life stages of spotted rose snapper: early (EJ, middle (MJ and late juvenile (LJ with corresponding average weights of 21.3 ± 2.6 g (3 months after hatching, MAH, 190 ± 4.4 g (7 MAH, and 400 ± 11.5 g (12 MAH. At sampling points, the digestive tract was dissected into the stomach (St, pyloric caeca (PC, and the intestine in three sections (proximal (PI, middle (MI and distal intestine (DI. The effect of pH and temperature and specific inhibitors were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead a better comprehension of changes in digestive potential of Lutjanidae fish.

  10. Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

    Science.gov (United States)

    Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun

    2012-12-21

    In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

    Science.gov (United States)

    Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

    2017-11-01

    An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Plasma uric acid and tumor volume are highly predictive of outcome in nasopharyngeal carcinoma patients receiving intensity modulated radiotherapy

    International Nuclear Information System (INIS)

    Lin, Hui; Lin, Huan-Xin; Ge, Nan; Wang, Hong-Zhi; Sun, Rui; Hu, Wei-Han

    2013-01-01

    The combined predictive value of plasma uric acid and primary tumor volume in nasopharyngeal carcinoma (NPC) patients receiving intensity modulated radiation therapy (IMRT) has not yet been determined. In this retrospective study, plasma uric acid level was measured after treatment in 130 histologically-proven NPC patients treated with IMRT. Tumor volume was calculated from treatment planning CT scans. Overall (OS), progression-free (PFS) and distant metastasis-free (DMFS) survival were compared using Kaplan-Meier analysis and the log rank test, and Cox multivariate and univariate regression models were created. Patients with a small tumor volume (<27 mL) had a significantly better DMFS, PFS and OS than patients with a large tumor volume. Patients with a high post-treatment plasma uric acid level (>301 μmol/L) had a better DMFS, PFS and OS than patients with a low post-treatment plasma uric acid level. Patients with a small tumor volume and high post-treatment plasma uric acid level had a favorable prognosis compared to patients with a large tumor volume and low post-treatment plasma uric acid level (7-year overall OS, 100% vs. 48.7%, P <0.001 and PFS, 100% vs. 69.5%, P <0.001). Post-treatment plasma uric acid level and pre-treatment tumor volume have predictive value for outcome in NPC patients receiving IMRT. NPC patients with a large tumor volume and low post-treatment plasma uric acid level may benefit from additional aggressive treatment after IMRT

  13. Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor.

    Science.gov (United States)

    Bastías, Adriana; López-Climent, María; Valcárcel, Mercedes; Rosello, Salvador; Gómez-Cadenas, Aurelio; Casaretto, José A

    2011-03-01

    Growing evidence suggests that the phytohormone abscisic acid (ABA) plays a role in fruit development. ABA signaling components of developmental programs and responses to stress conditions include the group of basic leucine zipper transcriptional activators known as ABA-response element binding factors (AREBs/ABFs). AREB transcription factors mediate ABA-regulated gene expression involved in desiccation tolerance and are expressed mainly in seeds and in vegetative tissues under stress; however, they are also expressed in some fruits such as tomato. In order to get an insight into the role of ABA signaling in fruit development, the expression of two AREB-like factors were investigated during different developmental stages. In addition, tomato transgenic lines that overexpress and downregulate one AREB-like transcription factor, SlAREB1, were used to determine its effect on the levels of some metabolites determining fruit quality. Higher levels of citric acid, malic acid, glutamic acid, glucose and fructose were observed in SlAREB1-overexpressing lines compared with those in antisense suppression lines in red mature fruit pericarp. The higher hexose concentration correlated with increased expression of genes encoding a vacuolar invertase (EC 3.2.1.26) and a sucrose synthase (EC 2.4.1.13). No significant changes were found in ethylene content which agrees with the normal ripening phenotype observed in transgenic fruits. These results suggest that an AREB-mediated ABA signal affects the metabolism of these compounds during the fruit developmental program. Copyright © Physiologia Plantarum 2010.

  14. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

    Directory of Open Access Journals (Sweden)

    Zongyun Chen

    Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

  15. Gut microbiota alterations and dietary modulation in childhood malnutrition - The role of short chain fatty acids

    DEFF Research Database (Denmark)

    Pekmez, Ceyda Tugba; Dragsted, Lars Ove; Brahe, Lena Kirchner

    2018-01-01

    and metabolism through enteroendocrine cell signaling, adipogenesis and insulin-like growth factor-1 production. Elucidating these mechanisms may lead to development of new modulation practices of the gut microbiota as a potential prevention and treatment strategy for childhood malnutrition. The present overview......, and the potential of probiotics, prebiotics and synbiotics for modulating the gut microbiota during childhood as a prevention and treatment strategy against undernutrition and obesity....

  16. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

  17. ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme

    Directory of Open Access Journals (Sweden)

    Marwa E. Elsherbiny

    2018-04-01

    Full Text Available Glioblastoma multiforme (GBM is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA to docosahexaenoic acid (DHA driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054 between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4% in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

  18. Microbiota-Derived Short-Chain Fatty Acids Modulate Expression of Campylobacter jejuni Determinants Required for Commensalism and Virulence.

    Science.gov (United States)

    Luethy, Paul M; Huynh, Steven; Ribardo, Deborah A; Winter, Sebastian E; Parker, Craig T; Hendrixson, David R

    2017-05-09

    Campylobacter jejuni promotes commensalism in the intestinal tracts of avian hosts and diarrheal disease in humans, yet components of intestinal environments recognized as spatial cues specific for different intestinal regions by the bacterium to initiate interactions in either host are mostly unknown. By analyzing a C. jejuni acetogenesis mutant defective in converting acetyl coenzyme A (Ac-CoA) to acetate and commensal colonization of young chicks, we discovered evidence for in vivo microbiota-derived short-chain fatty acids (SCFAs) and organic acids as cues recognized by C. jejuni that modulate expression of determinants required for commensalism. We identified a set of C. jejuni genes encoding catabolic enzymes and transport systems for amino acids required for in vivo growth whose expression was modulated by SCFAs. Transcription of these genes was reduced in the acetogenesis mutant but was restored upon supplementation with physiological concentrations of the SCFAs acetate and butyrate present in the lower intestinal tracts of avian and human hosts. Conversely, the organic acid lactate, which is abundant in the upper intestinal tract where C. jejuni colonizes less efficiently, reduced expression of these genes. We propose that microbiota-generated SCFAs and lactate are cues for C. jejuni to discriminate between different intestinal regions. Spatial gradients of these metabolites likely allow C. jejuni to locate preferred niches in the lower intestinal tract and induce expression of factors required for intestinal growth and commensal colonization. Our findings provide insights into the types of cues C. jejuni monitors in the avian host for commensalism and likely in humans to promote diarrheal disease. IMPORTANCE Campylobacter jejuni is a commensal of the intestinal tracts of avian species and other animals and a leading cause of diarrheal disease in humans. The types of cues sensed by C. jejuni to influence responses to promote commensalism or

  19. Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid, Capsule, and Surface Proteins.

    Directory of Open Access Journals (Sweden)

    Yoshiki Misawa

    2015-07-01

    Full Text Available Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA mutant (ΔtagO failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.

  20. Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

    International Nuclear Information System (INIS)

    Shen, W.; Fletcher, T.S.; Largman, C.

    1987-01-01

    Although protease E was isolated from human pancreas over 10 years ago, its amino acid sequence and relationship to the elastases have not been established. The authors report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. They have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. They also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1

  1. Modulation of cardiopulmonary depressor reflex in nucleus ambiguus by electroacupuncture: roles of opioids and γ-aminobutyric acid.

    Science.gov (United States)

    Tjen-A-Looi, Stephanie C; Li, Peng; Li, Min; Longhurst, John C

    2012-04-01

    Stimulation of cardiopulmonary receptors with phenylbiguanide (PBG) elicits depressor cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated in part by the brain stem parasympathetic cardiac neurons in the nucleus ambiguus (NAmb). The present study examined NAmb neurotransmitter mechanisms underlying the influence of electroacupuncture (EA) on the PBG-induced hypotension and bradycardia. We hypothesized that somatic stimulation during EA modulates PBG responses through opioid and γ-aminobutyric acid (GABA) modulation in the NAmb. Anesthetized and ventilated cats were studied during repeated stimulation with PBG or cardiac vagal afferents while low-frequency EA (2 Hz) was applied at P5-6 acupoints overlying the median nerve for 30 min and NAmb neuronal activity, heart rate, and blood pressure were recorded. Microinjection of kainic acid into the NAmb attenuated the PBG-induced bradycardia from -60 ± 11 to -36 ± 11 beats/min. Likewise, EA reduced the PBG-induced depressor and bradycardia reflex by 52 and 61%, respectively. Cardiac vagal afferent evoked preganglionic cellular activity in the NAmb was reduced by EA for about 60 min. Blockade of opioid or GABA(A) receptors using naloxone and gabazine reversed the EA-related modulation of the evoked cardiac vagal activity by 73 and 53%, respectively. Similarly, naloxone and gabazine reversed EA modulation of the negative chronotropic responses from -11 ± 5 to -23 ± 6 and -13 ± 4 to -24 ± 3 beats/min, respectively. Thus EA at P5-6 decreases PBG evoked hypotension and bradycardia as well as the NAmb PBG-sensitive preganglionic cardiac vagal outflow through opioid and GABA neurotransmitter systems.

  2. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

    Science.gov (United States)

    Lomate, Purushottam R.; Bonning, Bryony C.

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

  3. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

    Science.gov (United States)

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans

    2003-05-01

    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  4. Essential fatty acids and their metabolites as modulators of stem cell biology with reference to inflammation, cancer, and metastasis.

    Science.gov (United States)

    Das, Undurti N

    2011-12-01

    Stem cells are pluripotent and expected to be of benefit in the management of coronary heart disease, stroke, diabetes mellitus, cancer, and Alzheimer's disease in which pro-inflammatory cytokines are increased. Identifying endogenous bioactive molecules that have a regulatory role in stem cell survival, proliferation, and differentiation may aid in the use of stem cells in various diseases including cancer. Essential fatty acids form precursors to both pro- and anti-inflammatory molecules have been shown to regulate gene expression, enzyme activity, modulate inflammation and immune response, gluconeogenesis via direct and indirect pathways, function directly as agonists of a number of G protein-coupled receptors, activate phosphatidylinositol 3-kinase/Akt and p44/42 mitogen-activated protein kinases, and stimulate cell proliferation via Ca(2+), phospholipase C/protein kinase, events that are also necessary for stem cell survival, proliferation, and differentiation. Hence, it is likely that bioactive lipids play a significant role in various diseases by modulating the proliferation and differentiation of embryonic stem cells in addition to their capacity to suppress inflammation. Ephrin Bs and reelin, adhesion molecules, and microRNAs regulate neuronal migration and cancer cell metastasis. Polyunsaturated fatty acids and their products seem to modulate the expression of ephrin Bs and reelin and several adhesion molecules and microRNAs suggesting that bioactive lipids participate in neuronal regeneration and stem cell proliferation, migration, and cancer cell metastasis. Thus, there appears to be a close interaction among essential fatty acids, their bioactive products, and inflammation and cancer growth and its metastasis.

  5. Applications of electrochemically-modulated liquid chromatography (EMLC): Separations of aromatic amino acids and polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Li [Iowa State Univ., Ames, IA (United States)

    1998-03-27

    The research in this thesis explores the separation capabilities of a new technique termed electrochemically-modulated liquid chromatography (EMLC). The thesis begins with a general introduction section which provides a literature review of this technique as well as a brief background discussion of the two research projects in each of the next two chapters. The two papers which follow investigate the application of EMLC to the separation of a mixture of aromatic amino acids and of a mixture of polycyclic aromatic hydrocarbons (PAHs). The last section presents general conclusions and summarizes the thesis. References are compiled in the reference section of each chapter. The two papers have been removed for separate processing.

  6. Synthesis of Nickel (Ni) Doped HKUST-1 Using Solvotermal Method with Addition of Acetic Acid as Modulator

    OpenAIRE

    Safii, Farhan Fikri; Ediati, Ratna

    2015-01-01

    Hong Kong University of Science and Technology-1 (HKUST-1) is metal organic framework (MOF) that composed from ligand BTC (1,3,5-benzene tricarboxylic) and copper ions. The method used in this study is solvotermal with variations nickel ion doping and additions acetic acid as modulator. The purpose of this study is to increase the hydrogen storage capacity. The results obtained were characterized by XRD showed that the HKUST-1 and Ni-HKUST-1 has formed, showed by peaks at 2 theta = 6.7, 9.5, ...

  7. Bile acids modulate glucocorticoid metabolism and the hypothalamic-pituitary-adrenal axis in obstructive jaundice

    DEFF Research Database (Denmark)

    McNeilly, Alison D; Macfarlane, David P; O'Flaherty, Emmett

    2010-01-01

    Suppression of the hypothalamic-pituitary-adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5bet...

  8. Retinoic acid modulation of ultraviolet light-induced epidermal ornithine decarboxylase activity

    International Nuclear Information System (INIS)

    Lowe, N.J.; Breeding, J.

    1982-01-01

    Irradiation of skin with ultraviolet light of sunburn range (UVB) leads to a large and rapid induction of the polyamine biosynthetic enzyme ornithine decarboxylase in the epidermis. Induction of epidermal ornithine decarboxylase also occurs following application of the tumor promoting agent 12-0-tetradecanoylphorbol-13 acetate and topical retinoic acid is able to block both this ornithine decarboxylase induction and skin tumor promotion. In the studies described below, topical application of retinoic acid to hairless mouse skin leads to a significant inhibition of UVB-induced epidermal ornithine decarboxylase activity. The degree of this inhibition was dependent on the dose, timing, and frequency of the application of retinoic acid. To show significant inhibition of UVB-induced ornithine decarboxylase the retinoic acid had to be applied within 5 hr of UVB irradiation. If retinoic acid treatment was delayed beyond 7 hr following UVB, then no inhibition of UVB-induced ornithine decarboxylase was observed. The quantities of retinoic acid used (1.7 nmol and 3.4 nmol) have been shown effective at inhibiting 12-0-tetradecanoyl phorbol-13 acetate induced ornithine decarboxylase. The results show that these concentrations of topical retinoic acid applied either before or immediately following UVB irradiation reduces the UVB induction of epidermal ornithine decarboxylase. The effect of retinoic acid in these regimens on UVB-induced skin carcinogenesis is currently under study

  9. Modulation of the chicken immune cell function by dietary polyunsaturated fatty acids

    NARCIS (Netherlands)

    Sijben, J.W.C.

    2002-01-01

    Polyunsaturated fatty acids (PUFA) possess a wide range of biological properties, including immunomodulation. The amount, type, and ratio of dietary PUFA determine the types of fatty acids that are incorporated into immune cell membranes. Consequently, the physiological properties of immune cells

  10. Hop acid-rich spent craft brewer's yeast modulates gut bacterial growth

    Science.gov (United States)

    Alpha and beta hop acids (humulones and lupulones) from Humulus lupulus are inhibitors of Gram-positive organisms and important natural antibiotics for beer fermentation and carbohydrate feed stocks for biofuel production. Recent observations (Bryant and Cohen) of high levels of hop acids in spent ...

  11. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    Science.gov (United States)

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  12. Dietary omega-3 fatty acids aid in the modulation of inflammation and metabolic health

    Directory of Open Access Journals (Sweden)

    J. Bruce German

    2011-07-01

    Full Text Available This article focuses on the role of omega-3 fatty acids as precursors for lipid signaling molecules known as oxylipins. Although omega-3 fatty acids are beneficial in autoimmune disorders, inflammatory diseases and heart disease, they are generally underrepresented in the American diet. A literature review confirms that the consumption of omega-3 fatty acids - whether in food sources such as walnuts, flax seeds and fatty fish (including salmon and sardines, or in supplements - is associated with decreased morbidity and mortality. This growing body of evidence, including the results of a recent study of patients with kidney disease, highlights the need to measure omega-3 fatty acids and their oxylipin products as markers of metabolic health and biomarkers of disease. In addition, there is substantial evidence of the need to increase the omega-3 fatty acid content of American diets to optimize metabolic health.

  13. Cichorium intybus L. promotes intestinal uric acid excretion by modulating ABCG2 in experimental hyperuricemia.

    Science.gov (United States)

    Wang, Yu; Lin, Zhijian; Zhang, Bing; Nie, Anzheng; Bian, Meng

    2017-01-01

    Excessive production and/or reduced excretion of uric acid could lead to hyperuricemia, which could be a major cause of disability. Hyperuricemia has received increasing attention in the last few decades due to its global prevalence. Cichorium intybus L., commonly known as chicory, is a perennial herb of the asteraceae family. It was previously shown to exert potent hypouricemic effects linked with decreasing uric acid formation in the liver by down-regulating the activity of xanthine oxidase, and increasing uric acid excretion by up-regulating the renal OAT3 mRNA expression. The present study aimed to evaluate its extra-renal excretion and possible molecular mechanism underlying the transporter responsible for intestinal uric acid excretion in vivo. Chicory was administered intragastrically to hyperuricemic rats induced by drinking 10% fructose water. The uricosuric effect was evaluated by determining the serum uric acid level as well as the intestinal uric acid excretion by HPLC. The location and expression levels of ATP-binding cassette transporter, sub-family G, member 2 (ABCG2) in jejunum and ileum were analyzed. The administration of chicory decreased the serum uric acid level significantly and increased the intestinal uric acid excretion obviously in hyperuricemic rats induced by 10% fructose drinking. Staining showed that ABCG2 was expressed in the apical membrane of the epithelium and glands of the jejunum and ileum in rats. Further examination showed that chicory enhanced the mRNA and protein expressions of ABCG2 markedly in a dose-dependent manner in jejunum and ileum. These findings indicate that chicory increases uric acid excretion by intestines, which may be related to the stimulation of intestinal uric acid excretion via down-regulating the mRNA and protein expressions of ABCG2.

  14. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    International Nuclear Information System (INIS)

    Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.; Holan, G.

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [ 3 H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [ 3 H]diazepam binding are those that are active in displacing [ 3 H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed

  15. Docosahexaenoic acid (DHA) supplementation in pregnancy differentially modulates arachidonic acid and DHA status across FADS genotypes in pregnancy.

    Science.gov (United States)

    Scholtz, S A; Kerling, E H; Shaddy, D J; Li, S; Thodosoff, J M; Colombo, J; Carlson, S E

    2015-03-01

    Some FADS alleles are associated with lower DHA and ARA status assessed by the relative amount of arachidonic acid (ARA) and docosahexaenoic acid (DHA) in plasma and red blood cell (RBC) phospholipids (PL). We determined two FADS single nucleotide polymorphisms (SNPs) in a cohort of pregnant women and examined the relationship of FADS1rs174533 and FADS2rs174575 to DHA and ARA status before and after supplementation with 600mg per day of DHA. The 205 pregnant women studied were randomly assigned to placebo (mixed soy and corn oil) (n=96) or 600mg algal DHA (n=109) in 3 capsules per day for the last two trimesters of pregnancy. Women homozygous for the minor allele of FADS1rs174533 (but not FADS2rs174575) had lower DHA and ARA status at baseline. At delivery, minor allele homozygotes of FADS1rs174533 in the placebo group had lower RBC-DHA compared to major-allele carriers (P=0.031), while in the DHA-supplemented group, all genotypes had higher DHA status compared to baseline (P=0.001) and status did not differ by genotype (P=0.941). Surprisingly, DHA but not the placebo decreased ARA status of minor allele homozygotes of both FADS SNPs but not major allele homozygotes at delivery. Any physiological effects of changing the DHA to ARA ratio by increasing DHA intake appears to be greater in minor allele homozygotes of some FADS SNPs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

    Directory of Open Access Journals (Sweden)

    Yu-Yin Li

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

  17. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  18. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Science.gov (United States)

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-08

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

  19. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

    Science.gov (United States)

    Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

    1992-08-01

    The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

  20. A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus.

    Science.gov (United States)

    Moon, S H; Parulekar, S J

    1991-03-05

    Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design

  1. Adipose Tissue Branched Chain Amino Acid (BCAA) Metabolism Modulates Circulating BCAA Levels*

    OpenAIRE

    Herman, Mark A.; She, Pengxiang; Peroni, Odile D.; Lynch, Christopher J.; Kahn, Barbara B.

    2010-01-01

    Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent obse...

  2. C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

    Science.gov (United States)

    Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

    2004-01-30

    C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

  3. Maternal omega-3 fatty acids regulate offspring obesity through persistent modulation of gut microbiota.

    Science.gov (United States)

    Robertson, Ruairi C; Kaliannan, Kanakaraju; Strain, Conall R; Ross, R Paul; Stanton, Catherine; Kang, Jing X

    2018-05-24

    The early-life gut microbiota plays a critical role in host metabolism in later life. However, little is known about how the fatty acid profile of the maternal diet during gestation and lactation influences the development of the offspring gut microbiota and subsequent metabolic health outcomes. Here, using a unique transgenic model, we report that maternal endogenous n-3 polyunsaturated fatty acid (PUFA) production during gestation or lactation significantly reduces weight gain and markers of metabolic disruption in male murine offspring fed a high-fat diet. However, maternal fatty acid status appeared to have no significant effect on weight gain in female offspring. The metabolic phenotypes in male offspring appeared to be mediated by comprehensive restructuring of gut microbiota composition. Reduced maternal n-3 PUFA exposure led to significantly depleted Epsilonproteobacteria, Bacteroides, and Akkermansia and higher relative abundance of Clostridia. Interestingly, offspring metabolism and microbiota composition were more profoundly influenced by the maternal fatty acid profile during lactation than in utero. Furthermore, the maternal fatty acid profile appeared to have a long-lasting effect on offspring microbiota composition and function that persisted into adulthood after life-long high-fat diet feeding. Our data provide novel evidence that weight gain and metabolic dysfunction in adulthood is mediated by maternal fatty acid status through long-lasting restructuring of the gut microbiota. These results have important implications for understanding the interaction between modern Western diets, metabolic health, and the intestinal microbiome.

  4. Higher acclimation temperature modulates the composition of muscle fatty acid of Tor putitora juveniles

    Directory of Open Access Journals (Sweden)

    M.S. Akhtar

    2014-08-01

    Full Text Available A 30-day acclimation trial was conducted using golden mahseer, Tor putitora juveniles to study its muscle fatty acid composition at five acclimation temperatures (AT. Ninety juveniles of T. putitora were distributed among five treatment groups (20, 23, 26, 29 and 32±0.5 °C. At the end of 30 days trial, highest percentage of monounsaturated fatty acids was found at 20 °C and lowest at 26 °C. The highest percentage of n-6 polyunsaturated fatty acid (PUFA was found at 23 °C and a decreasing trend was observed with increase in AT. However, highest percentage of n-3 PUFA was found at 32 °C and lowest at 29 °C. The maximum n-6 to n-3 ratio was observed at 23 °C and ratio decreased to a minimum at 32 °C. The results revealed that T. putitora juveniles could adapt to higher acclimation temperatures by altering its muscle fatty acid composition mainly by increasing its total saturated fatty acids especially stearic acid.

  5. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  6. EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

    Science.gov (United States)

    Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

    2017-03-01

    The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

  7. Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

    Science.gov (United States)

    Li, Ming-Hua; Leng, Tian-Dong; Feng, Xue-Chao; Yang, Tao; Simon, Roger P; Xiong, Zhi-Gang

    2016-08-26

    An important contributor to brain ischemia is known to be extracellular acidosis, which activates acid-sensing ion channels (ASICs), a family of proton-gated sodium channels. Lines of evidence suggest that targeting ASICs may lead to novel therapeutic strategies for stroke. Investigations of the role of ASICs in ischemic brain injury have naturally focused on the role of extracellular pH in ASIC activation. By contrast, intracellular pH (pHi) has received little attention. This is a significant gap in our understanding because the ASIC response to extracellular pH is modulated by pHi, and activation of ASICs by extracellular protons is paradoxically enhanced by intracellular alkalosis. Our previous studies show that acidosis-induced cell injury in in vitro models is attenuated by intracellular acidification. However, whether pHi affects ischemic brain injury in vivo is completely unknown. Furthermore, whereas ASICs in native neurons are composed of different subunits characterized by distinct electrophysiological/pharmacological properties, the subunit-dependent modulation of ASIC activity by pHi has not been investigated. Using a combination of in vitro and in vivo ischemic brain injury models, electrophysiological, biochemical, and molecular biological approaches, we show that the intracellular alkalizing agent quinine potentiates, whereas the intracellular acidifying agent propionate inhibits, oxygen-glucose deprivation-induced cell injury in vitro and brain ischemia-induced infarct volume in vivo Moreover, we find that the potentiation of ASICs by quinine depends on the presence of the ASIC1a, ASIC2a subunits, but not ASIC1b, ASIC3 subunits. Furthermore, we have determined the amino acids in ASIC1a that are involved in the modulation of ASICs by pHi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Fatty acids isolated from royal jelly modulate dendritic cell-mediated immune response in vitro.

    Science.gov (United States)

    Vucevic, Dragana; Melliou, Eleni; Vasilijic, Sasa; Gasic, Sonja; Ivanovski, Petar; Chinou, Ioanna; Colic, Miodrag

    2007-09-01

    Royal jelly (RJ), especially its protein components, has been shown to possess immunomodulatory activity. However, almost nothing is known about the influence of RJ fatty acids on the immune system. In this work we studied the effect of 10-hydroxy-2-decanoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10-DDA), isolated from RJ, on the immune response using a model of rat dendritic cell (DC)-T-cell cocultures. Both fatty acids, at higher concentrations, inhibited the proliferation of allogeneic T cells. The effect of 10-HDA was stronger and was followed by a decrease in interleukin-2 (IL-2) production and down-regulation of IL-2 receptor expression. Spleen DC, cultivated with 10 microg/ml of fatty acids down-regulated the expression of CD86 and the production of IL-12, but up-regulated the production of IL-10. In contrast, DC, pretreated with 100 microg/ml of 3,10-DDA, up-regulated the expression of CD86 and augmented the proliferation of allogeneic T cells. The highest dose (200 microg/ml) of both fatty acids which was non-apoptotic for both T cells and DC, down-regulated the expression of MHC class II and CD86, decreased the production of IL-12 and made these DC less allostimulatory. The immunosuppressive activity of 3,10-DDA was also confirmed in vivo, using a model of Keyhole lymphet hemocyanine immunization of rats. In conclusion, our results showed the immunomodulatory activity of RJ fatty acids and suggest that DC are a significant target of their action.

  9. Preconception folic acid use modulates estradiol and follicular responses to ovarian stimulation.

    Science.gov (United States)

    Twigt, John M; Hammiche, Fatima; Sinclair, Kevin D; Beckers, Nicole G; Visser, Jenny A; Lindemans, Jan; de Jong, Frank H; Laven, Joop S E; Steegers-Theunissen, Régine P

    2011-02-01

    Folate is a methyl donor. Availability of folate affects DNA methylation profiles and thereby gene expression profiles. We investigated the effects of low-dose folic acid use (0.4 mg/d) on the ovarian response to mild and conventional ovarian stimulation in women. In a randomized trial among subfertile women, 24 and 26 subjects received conventional and mild ovarian stimulation, respectively. Blood samples were taken during the early follicular phase of the cycle prior to treatment and on the day of human chorionic gonadotropin administration for determination of serum total homocysteine, anti-Müllerian hormone (AMH), estradiol, and folate. Folic acid use was validated by questionnaire and serum folate levels. Preovulatory follicles were visualized, counted, and diameters recorded using transvaginal ultrasound. The relation between folic acid use and ovarian response was assessed using linear regression analysis. Folic acid use modified the ovarian response to ovarian stimulation treatment. The estradiol response was higher in nonfolic acid users receiving conventional treatment [β(interaction) = 0.52 (0.07-0.97); P = 0.03], and this effect was independent of serum AMH levels and the preovulatory follicle count. In the conventional treatment, the mean follicle number was also greater in nonusers compared with the users group (14.1 vs. 8.9, P = 0.03). Low-dose folic acid use attenuates follicular and endocrine responses to conventional stimulation, independent of AMH and follicle count. The nature of this observation suggests that the effect of folic acid is most prominent during early follicle development, affecting immature follicles. Deleterious effects of folate deficiency, like DNA hypomethylation and oxidative stress, can help to explain our observations.

  10. Pathogen-secreted proteases activate a novel plant immune pathway.

    Science.gov (United States)

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  11. Modulation of fatty acid composition and growth in Sporosarcina species in response to temperatures and exogenous branched-chain amino acids.

    Science.gov (United States)

    Tsuda, Kentaro; Nagano, Hideaki; Ando, Akinori; Shima, Jun; Ogawa, Jun

    2017-06-01

    Psychrotolerant endospore-forming Sporosarcina species have been predominantly isolated from minced fish meat (surimi), which is stored under refrigeration after heat treatment. To develop a better method for preserving surimi-based food products, we studied the growth and fatty acid compositions of the isolated strain S92h as well as Sporosarcina koreensis and Sporosarcina aquimarina at cold and moderate temperatures. The growth rates of strain S92h and S. koreensis were the fastest and slowest at cold temperatures, respectively, although these strains grew at a similar rate at moderate temperatures. In all three strains, the proportions of anteiso-C 15:0 and unsaturated fatty acids (UFAs) were significantly higher at cold temperatures than at moderate temperatures. Furthermore, supplementation with valine, leucine, and isoleucine resulted in proportional increases in iso-C 16:0 , iso-C 15:0 , and anteiso-C 15:0 , respectively, among the fatty acid compositions of these strains. The proportions of the UFAs were also altered by the supplementation. At cold temperatures, the growth rates of strain S92h and S. koreensis, but not of S. aquimarina, were affected by supplementation with leucine. Supplementation with isoleucine enhanced the growth of S. koreensis at cold temperatures but not that of the other strains. Valine did not affect the growth of any strain. These results indicate that anteiso-C 15:0 and UFAs both play important roles in the cold tolerance of the genus Sporosarcina and that these bacteria modulate their fatty acid compositions in response to the growth environment.

  12. Differential responses of sugar, organic acids and anthocyanins to source-sink modulation in Cabernet Sauvignon and Sangiovese grapevines.

    Science.gov (United States)

    Bobeica, Natalia; Poni, Stefano; Hilbert, Ghislaine; Renaud, Christel; Gomès, Eric; Delrot, Serge; Dai, Zhanwu

    2015-01-01

    Grape berry composition mainly consists of primary and secondary metabolites. Both are sensitive to environment and viticultural management. As a consequence, climate change can affect berry composition and modify wine quality and typicity. Leaf removal techniques can impact berry composition by modulating the source-to-sink balance and, in turn, may mitigate some undesired effects due to climate change. The present study investigated the balance between technological maturity parameters such as sugars and organic acids, and phenolic maturity parameters such as anthocyanins in response to source-sink modulation. Sugar, organic acid, and anthocyanin profiles were compared under two contrasting carbon supply levels in berries of cv. Cabernet Sauvignon and Sangiovese collected at 9 and 14 developmental stages respectively. In addition, whole-canopy net carbon exchange rate was monitored for Sangiovese vines and a mathematic model was used to calculate the balance between carbon fixation and berry sugar accumulation. Carbon limitation affected neither berry size nor the concentration of organic acids at harvest. However, it significantly reduced the accumulation of sugars and total anthocyanins in both cultivars. Most interestingly, carbon limitation decreased total anthocyanin concentration by 84.3% as compared to the non source-limited control, whereas it decreased sugar concentration only by 27.1%. This suggests that carbon limitation led to a strong imbalance between sugars and anthocyanins. Moreover, carbon limitation affected anthocyanin profiles in a cultivar dependent manner. Mathematical analysis of carbon-balance indicated that berries used a higher proportion of fixed carbon for sugar accumulation under carbon limitation (76.9%) than under carbon sufficiency (48%). Thus, under carbon limitation, the grape berry can manage the metabolic fate of carbon in such a way that sugar accumulation is maintained at the expense of secondary metabolites.

  13. Differential responses of sugar, organic acids and anthocyanins to source-sink modulation in Cabernet Sauvignon and Sangiovese grapevines

    Directory of Open Access Journals (Sweden)

    Natalia eBobeica

    2015-05-01

    Full Text Available Grape berry composition mainly consists of primary and secondary metabolites. Both are sensitive to environment and viticultural management. As a consequence, climate change can affect berry composition and modify wine quality and typicity. Leaf removal techniques can impact berry composition by modulating the source-to-sink balance and, in turn, may mitigate some undesired effects due to climate change. The present study investigated the balance between technological maturity parameters such as sugars and organic acids, and phenolic maturity parameters such as anthocyanins in response to source-sink modulation. Sugar, organic acid, and anthocyanin profiles were compared under two contrasting carbon supply levels in berries of cv. Cabernet Sauvignon and Sangiovese collected at 9 and 14 developmental stages respectively. In addition, whole-canopy net carbon exchange rate was monitored for Sangiovese vines and a mathematic model was used to calculate the balance between carbon fixation and berry sugar accumulation. Carbon limitation affected neither berry size nor the concentration of organic acids at harvest. However, it significantly reduced the accumulation of sugars and total anthocyanins in both cultivars. Most interestingly, carbon limitation decreased total anthocyanin concentration by 84.3 % as compared to the non source-limited control, whereas it decreased sugar concentration only by 27.1 %. This suggests that carbon limitation led to a strong imbalance between sugars and anthocyanins. Moreover, carbon limitation affected anthocyanin profiles in a cultivar dependent manner. Mathematical analysis of carbon-balance indicated that berries used a higher proportion of fixed carbon for sugar accumulation under carbon limitation (76.9% than under carbon sufficiency (48%. Thus, under carbon limitation, the grape berry can manage the metabolic fate of carbon in such a way that sugar accumulation is maintained at the expense of secondary

  14. Epoxy fatty acids and inhibition of the soluble epoxide hydrolase selectively modulate GABA mediated neurotransmission to delay onset of seizures.

    Directory of Open Access Journals (Sweden)

    Bora Inceoglu

    Full Text Available In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA. ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs. The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH, the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.

  15. Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion.

    Science.gov (United States)

    Silva, Jerson L; Vieira, Tuane C R G; Gomes, Mariana P B; Rangel, Luciana P; Scapin, Sandra M N; Cordeiro, Yraima

    2011-03-01

    The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin-αv trafficking.

    Science.gov (United States)

    Prichard, David O; Byrne, Anne Marie; Murphy, James O; Reynolds, John V; O'Sullivan, Jacintha; Feighery, Ronan; Doyle, Brendan; Eldin, Osama Sharaf; Finn, Stephen P; Maguire, Aoife; Duff, Deirdre; Kelleher, Dermot P; Long, Aideen

    2017-12-01

    The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET-1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α 3 , α 4, α 5 , α 6 and α ν ). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET-1A cells to vitronectin and reduced cell-surface expression of integrin-α ν via effects on endocytic recycling processes. Increased expression of integrin-α v was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin-α ν was observed in QH BO cells compared to HET-1A cells. QH cells were resistant to DCA-mediated loss of adhesion and reduction in cell-surface expression of integrin-α ν . We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin α ν expression, providing a novel mechanism for bile acid-mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid-mediated erosion should be considered in the clinical management of patients with GORD. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Ethanolic extract of Piper betle Linn. leaves reduces nociception via modulation of arachidonic acid pathway.

    Science.gov (United States)

    De, Soumita; Maroo, Niteeka; Saha, Piu; Hazra, Samik; Chatterjee, Mitali

    2013-01-01

    The objective of this study was to evaluate the peripheral analgesic effect of Piper betle leaf extract (PBE) along with establishing its putative mechanism of action. Male Swiss albino mice after pre-treatment (1 h) with different doses of PBE were injected 0.8% (v/v) acetic acid i.p.; the onset and number of writhes were noted up to 15 min. To evaluate the mechanism of action, the murine peritoneal exudate was incubated with PBE for 1 h, followed by exposure to arachidonic acid (AA) and generation of reactive oxygen species (ROS) was measured by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate. PBE in a dose dependent manner significantly reduced acetic acid induced writhing response in mice (P < 0.001). In peritoneal exudates, PBE significantly inhibited AA induced generation of ROS, P < 0.01. The present study indicates that PBE has promising analgesic activity, worthy of future pharmacological consideration.

  18. Coordinations between gene modules control the operation of plant amino acid metabolic networks

    Directory of Open Access Journals (Sweden)

    Galili Gad

    2009-01-01

    Full Text Available Abstract Background Being sessile organisms, plants should adjust their metabolism to dynamic changes in their environment. Such adjustments need particular coordination in branched metabolic networks in which a given metabolite can be converted into multiple other metabolites via different enzymatic chains. In the present report, we developed a novel "Gene Coordination" bioinformatics approach and use it to elucidate adjustable transcriptional interactions of two branched amino acid metabolic networks in plants in response to environmental stresses, using publicly available microarray results. Results Using our "Gene Coordination" approach, we have identified in Arabidopsis plants two oppositely regulated groups of "highly coordinated" genes within the branched Asp-family network of Arabidopsis plants, which metabolizes the amino acids Lys, Met, Thr, Ile and Gly, as well as a single group of "highly coordinated" genes within the branched aromatic amino acid metabolic network, which metabolizes the amino acids Trp, Phe and Tyr. These genes possess highly coordinated adjustable negative and positive expression responses to various stress cues, which apparently regulate adjustable metabolic shifts between competing branches of these networks. We also provide evidence implying that these highly coordinated genes are central to impose intra- and inter-network interactions between the Asp-family and aromatic amino acid metabolic networks as well as differential system interactions with other growth promoting and stress-associated genome-wide genes. Conclusion Our novel Gene Coordination elucidates that branched amino acid metabolic networks in plants are regulated by specific groups of highly coordinated genes that possess adjustable intra-network, inter-network and genome-wide transcriptional interactions. We also hypothesize that such transcriptional interactions enable regulatory metabolic adjustments needed for adaptation to the stresses.

  19. Arachidonic acid-and docosahexaenoic acid-enriched formulas modulate antigen-specific T cell responses to influenza virus in neonatal piglets.

    Science.gov (United States)

    Bassaganya-Riera, Josep; Guri, Amir J; Noble, Alexis M; Reynolds, Kathryn A; King, Jennifer; Wood, Cynthia M; Ashby, Michael; Rai, Deshanie; Hontecillas, Raquel

    2007-03-01

    Whereas the immunomodulatory effects of feeding either arachidonic acid (AA) or docosahexaenoic acid (DHA) separately have been previously investigated, little is known about the immunomodulatory efficacy of AA or DHA when they are fed in combination as infant formula ingredients. The objective of this study was to investigate the ability of AA- and DHA(AA/DHA)-enriched infant formula to modulate immune responses in the neonate in response to an inactivated influenza virus vaccine. Neonatal piglets (n = 48) were weaned on day 2 of age and distributed into 16 blocks of 3 littermate piglets each. Within each block, piglets were randomly assigned to a control formula, AA/DHA-enriched formula (0.63% AA and 0.34% DHA), or sow milk for 30 d. On day 9, 8 blocks of piglets were immunized with an inactivated influenza virus vaccine. On days 0, 9, 16, 23, and 30 after weaning, we measured influenza virus-specific T cell proliferation and phenotype of T subsets in peripheral blood. A delayed-type hypersensitivity reaction test was administered on day 28. Cytokine messenger RNA expression was determined by quantitative real time reverse transcriptase-polymerase chain reaction on day 30. The influenza virus-specific CD4(+) and CD8(+) T cell ex vivo lymphoproliferative responses were significantly lower on day 23 after immunization in piglets receiving dietary AA/DHA supplementation and sow milk than in those receiving the unsupplemented control formula. The immunomodulatory effects of AA/DHA-enriched formulas were consistent with up-regulation of interleukin 10 in peripheral blood mononuclear cells. Overall, it appears that the AA/DHA-enriched formula modulated antigen-specific T cell responses in part through an interleukin 10-dependent mechanism.

  20. Structural changes in halophilic and non-halophilic proteases in response to chaotropic reagents.

    Science.gov (United States)

    Sinha, Rajeshwari; Khare, S K

    2014-08-01

    Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein-urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca(2+) and Na(+) ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.

  1. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protease production was 37°C at pH 9, with 2% inoculum in the medium for 24 h. .... Positive. Catalase test. Positive ... The enzyme activity gradually decreases from ... Effect of temperature on protease production by Pseudomonas fluorescens. 0 .... between RNA polymerase and upstream promotes DNA.

  2. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

    Pokorná, Jana; Machala, L.; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    Roč. 1, č. 3 (2009), s. 1209-1239 ISSN 1999-4915 R&D Projects: GA MŠk 1M0508 Grant - others:GA AV ČR(CZ) IAAX00320901 Program:IA Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * protease inhibitor * HAART Subject RIV: CE - Biochemistry

  3. Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid.

    Science.gov (United States)

    Hall, Susan; Anoopkumar-Dukie, Shailendra; Grant, Gary D; Desbrow, Ben; Lai, Richard; Arora, Devinder; Hong, Yinna

    2017-06-01

    Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100 nM and 10 μM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10 μM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10 nM and 10 μM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1 μM and 10 μM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.

  4. A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

    Science.gov (United States)

    Godahewa, G I; Perera, N C N; Lee, Sukkyoung; Kim, Myoung-Jin; Lee, Jehee

    2017-09-05

    Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone. Copyright © 2017. Published by Elsevier

  5. Maslinic acid modulates secreted phospholipase A2-IIA (sPLA2-IIA ...

    Indian Academy of Sciences (India)

    Wei Hsum Yap

    2018-03-29

    Mar 29, 2018 ... Further analysis revealed that sPLA2-IIA only induced modest LDL oxidation and that inhibitory .... COX-2, was also reduced in primary human chondrocyte, primary rat .... oxidation (4.34 nmol MDA/mg protein) compared to native ..... rheumatoid fibroblast-like synoviocyte arachidonic acid meta- bolism.

  6. How cocrystals of weakly basic drugs and acidic coformers might modulate solubility and stability.

    Science.gov (United States)

    Kuminek, G; Rodríguez-Hornedo, N; Siedler, S; Rocha, H V A; Cuffini, S L; Cardoso, S G

    2016-04-30

    Cocrystals of a weakly basic drug (nevirapine) with acidic coformers are shown to alter the solubility dependence on pH, and to exhibit a pHmax above which a less soluble cocrystal becomes more soluble than the drug. The cocrystal solubility advantage can be dialed up or down by solution pH.

  7. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B

    2000-01-01

    Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation...

  8. The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response on plant tissues and its action is modulated by salicylic acid and jasmonic acid signaling.

    Science.gov (United States)

    Rossi, Franco Rubén; Gárriz, Andrés; Marina, María; Romero, Fernando Matías; Gonzalez, María Elisa; Collado, Isidro González; Pieckenstain, Fernando Luis

    2011-08-01

    Botrytis cinerea, as a necrotrophic fungus, kills host tissues and feeds on the remains. This fungus is able to induce the hypersensitive response (HR) on its hosts, thus taking advantage on the host's defense machinery for generating necrotic tissues. However, the identity of HR effectors produced by B. cinerea is not clear. The aim of this work was to determine whether botrydial, a phytotoxic sesquiterpene produced by B. cinerea, is able to induce the HR on plant hosts, using Arabidopsis thaliana as a model. Botrydial induced the expression of the HR marker HSR3, callose deposition, and the accumulation of reactive oxygen species and phenolic compounds. Botrydial also induced the expression of PR1 and PDF1.2, two pathogenesis-related proteins involved in defense responses regulated by salicylic acid (SA) and jasmonic acid (JA), respectively. A. thaliana and tobacco plants defective in SA signaling were more resistant to botrydial than wild-type plants, as opposed to A. thaliana plants defective in JA signaling, which were more sensitive. It can be concluded that botrydial induces the HR on its hosts and its effects are modulated by host signaling pathways mediated by SA and JA.

  9. Neutrophil elastase inhibitor, ONO-5046, modulates acid-induced lung and systemic injury in rabbits.

    Science.gov (United States)

    Kaneko, K; Kudoh, I; Hattori, S; Yamada, H; Ohara, M; Wiener-Kronish, J; Okumura, F

    1997-09-01

    Acid instillation leads to direct lung and to secondary systemic organ injury, probably via activated macrophages and neutrophils. This study investigated the effects of neutrophil elastase on organ injury after unilateral lung acid instillation by administrating a specific neutrophil elastase inhibitor, ONO-5046, before acid instillation. Three groups of anesthetized rabbits (n = 12 in each group) underwent tracheostomies, and instillations were made into their right lower lobe airspaces with either phosphate buffered saline (pH, 7.4; volume, 1.2 ml/kg; n = 12) or HCl (pH, 1.25; volume, 1.2 ml/kg; n = 24). In half of the acid-instilled rabbits, ONO-5046, 10 mg/kg, was given intravenously 15 min before the HCl instillation, and then 10 mg x kg(-1) x h(-1) of the drug was continuously infused throughout the experiment. The other groups of animals received the vehicle intravenously. Anesthesia and mechanical ventilation was continued for 8 h, whereas arterial blood gases were sampled intermittently. Eight hours after saline or acid instillation, the animals were killed, and their lungs, heart, kidneys, liver, and small intestines were harvested. Wet-to-dry weight ratios (W/ D) and myeloperoxidase (MPO) assays of these organs were done, and elastase assays on the bronchoalveolar lavage fluids (BALF) obtained from each lung also were performed. Pretreatment with ONO-5046 attenuated the physiologic changes seen in the vehicle-treated animals. Significant decreases in W/D of the noninstilled lungs and of the small intestine and normalization of the oxygenation of the experimental animals occurred. The ONO-5046 pretreatment did not affect the neutrophil sequestration in the lungs or in the other organs as determined by neutrophil counts in BALF and by the MPO assays. A neutrophil elastase inhibitor, ONO-5046, administered immediately before acid instillation attenuated the physiologic changes seen in the vehicle-treated animals. The drug blocked neutrophil elastase but

  10. Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

    Directory of Open Access Journals (Sweden)

    Zhou Xiangdong

    2008-05-01

    Full Text Available Abstract Presenilin (PS1 or PS2 is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP. PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt, and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.

  11. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    Science.gov (United States)

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  12. Single-crystal Au microflakes modulated by amino acids and their sensing and catalytic properties.

    Science.gov (United States)

    Li, Mingjie; Wu, Xiaochen; Zhou, Jiyu; Kong, Qingshan; Li, Chaoxu

    2016-04-01

    Single-crystal Au microflakes with the planar area over 10(3)μm(2) (i.e. being accessible to the human eye resolution) were synthesized in an environment-friendly route by directing two-dimensional growth of Au nanocrystals into macroscopic scales with amino acids as both reducing agents and capping agents. Side groups of amino acids were found to be a determinant parameter to tune the dimension and size of Au single crystals. The successful synthesis of Au microflakes provides an unprecedented opportunity to bridge nanotechnology and macroscopic devices, and hereby to start a new scenario of exploring their unique properties and applications in optoelectronic devices and bio-sensing fields across multiple length scales. For example, Au microflakes respond to air humidity upon depositing on films of chitin nanofibrils, and sense various physiological molecules as electrode materials of biosensors. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

    Science.gov (United States)

    Tweeten, K A; Bulla, L A; Consigli, R A

    1978-06-01

    An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

  14. Production, Partial Purification and Characterization of Protease From Irradiated Streptomyces Spp

    International Nuclear Information System (INIS)

    Botros, H.W.; Ahmed, A.S.

    2011-01-01

    Production and partial purification of protease by the irradiated Streptomyces spp. was the aim of this study. Streptomyces spp. was allowed to grow in culture broth of 4% shrimp shells for purpose of inducing protease enzymes. Optimal conditions for protease production were 30 degree C, 0.3 kGy, ph 7, 5x10 4 /ml inoculum size and 7 days incubation period. Protease was purified by 80% ammonium sulphate saturation which exhibited 8.7 U/ml enzyme activity. Column chromatography using sephadex G-200 exerted 23.3 U/ml enzyme activity from pooled fraction (13-16). The molecular mass of protease was determined to be 39 kDa by SDS-PAGE. The enzyme was more stable over a wide range of ph 6-8 and temperature up to 40 degree C. The produced protease was activated by Ca, Mn and FeCl 2 and completely inhibited by ethylene-diamin tetraacetic acid (EDTA) at concentration of 1000 μg/ml

  15. Interactions between dietary oil treatments and genetic variants modulate fatty acid ethanolamides in plasma and body weight composition.

    Science.gov (United States)

    Pu, Shuaihua; Eck, Peter; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Jones, Peter J H

    2016-03-28

    Fatty acid ethanolamides (FAE), a group of lipid mediators derived from long-chain fatty acids (FA), mediate biological activities including activation of cannabinoid receptors, stimulation of fat oxidation and regulation of satiety. However, how circulating FAE levels are influenced by FA intake in humans remains unclear. The objective of the present study was to investigate the response of six major circulating FAE to various dietary oil treatments in a five-period, cross-over, randomised, double-blind, clinical study in volunteers with abdominal obesity. The treatment oils (60 g/12 552 kJ per d (60 g/3000 kcal per d)) provided for 30 d were as follows: conventional canola oil, high oleic canola oil, high oleic canola oil enriched with DHA, flax/safflower oil blend and corn/safflower oil blend. Two SNP associated with FAE degradation and synthesis were studied. Post-treatment results showed overall that plasma FAE levels were modulated by dietary FA and were positively correlated with corresponding plasma FA levels; minor allele (A) carriers of SNP rs324420 in gene fatty acid amide hydrolase produced higher circulating oleoylethanolamide (OEA) (P=0·0209) and docosahexaenoylethanolamide (DHEA) levels (P=0·0002). In addition, elevated plasma DHEA levels in response to DHA intake tended to be associated with lower plasma OEA levels and an increased gynoid fat mass. In summary, data suggest that the metabolic and physiological responses to dietary FA may be influenced via circulating FAE. Genetic analysis of rs324420 might help identify a sub-population that appears to benefit from increased consumption of DHA and oleic acid.

  16. Modulation of GABA receptors expressed in Xenopus oocytes by 13-L-hydroxylinoleic acid and food additives.

    Science.gov (United States)

    Aoshima, H; Tenpaku, Y

    1997-12-01

    To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on gamma-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 microM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

  17. Dietary fatty acids specifically modulate phospholipid pattern in colon cells with distinct differentiation capacities

    Czech Academy of Sciences Publication Activity Database

    Hofmanová, Jiřina; Slavík, J.; Ovesná, P.; Tylichová, Zuzana; Vondráček, Jan; Straková, Nicol; Vaculová, Alena; Cigánek, M.; Kozubík, Alois; Knopfová, L.; Šmarda, J.; Machala, M.

    2017-01-01

    Roč. 56, č. 4 (2017), s. 1493-1508 ISSN 1436-6207 R&D Projects: GA ČR GA13-09766S; GA MZd(CZ) NV15-30585A Institutional support: RVO:68081707 Keywords : docosahexaenoic acid * cancer-cells * epithelial-cells Subject RIV: FD - Oncology ; Hematology OBOR OECD: Oncology Impact factor: 4.370, year: 2016

  18. Modulation of mitomycin C-induced genotoxicity by acetyl- and thio- analogues of salicylic acid.

    Science.gov (United States)

    Pawar, Amol Ashok; Vikram, Ajit; Tripathi, Durga Nand; Padmanabhan, Shweta; Ramarao, Poduri; Jena, Gopabandhu

    2009-01-01

    Recent reports regarding acetylsalicylic acid (ASA) and its metabolites suggest suppressive effects against mitomycin C (MMC)-induced genotoxicity in a mice chromosomal aberration assay. Keeping this in mind, the potential anti-genotoxic effect of the thio-analogue of salicylic acid namely thio-salicylic acid (TSA) was speculated upon. The present study investigated and compared the anti-genotoxic potential of ASA and TSA. The study was performed in male swiss mice (20+/-2 g) using single-cell gel electrophoresis and a peripheral blood micronucleus assay. ASA and TSA (5, 10 or 20 mg/kg) were administered 15 minutes after MMC (1 mg/kg) once daily for 3 or 7 days. Both ASA and TSA significantly decreased the DNA damage induced by MMC as indicated by a decrease in the comet parameters in bone marrow cells and decreased frequencies of micronucleated reticulocytes in peripheral blood. The results clearly demonstrate the anti-genotoxic potential of ASA and TSA.

  19. Cutting back on the essentials: Can manipulating intake of specific amino acids modulate health and lifespan?

    Science.gov (United States)

    Brown-Borg, Holly M; Buffenstein, Rochelle

    2017-10-01

    With few exceptions, nutritional and dietary interventions generally impact upon both old-age quality of life and longevity. The life prolonging effects, commonly observed with dietary restriction reportedly are linked to alterations in protein intake and specifically limiting the dietary intake of certain essential amino acids. There is however a paucity of data methodically evaluating the various essential amino acids on health- and lifespan and the mechanisms involved. Rodent diets containing either lower methionine content, or tryptophan, than that found in commercially available chow, appear to elicit beneficial effects. It is unclear whether all of these favorable effects associated with restricted intake of methionine and tryptophan are due to their specific unique properties or if restriction of other essential amino acids, or proteins in general, may produce similar results. Considerably more work remains to be done to elucidate the mechanisms by which limiting these vital molecules may delay the onset of age-associated diseases and improve quality of life at older ages. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling.

    Science.gov (United States)

    Caires, Rebeca; Sierra-Valdez, Francisco J; Millet, Jonathan R M; Herwig, Joshua D; Roan, Esra; Vásquez, Valeria; Cordero-Morales, Julio F

    2017-10-03

    Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid's epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus Onygena corvina

    DEFF Research Database (Denmark)

    Huang, Yuhong; Kamp Busk, Peter; Herbst, Florian Alexander

    2015-01-01

    , the proteases secreted by O. corvina are interesting in view of their potential relevance for industrial decomposition of keratinaceous wastes. We sequenced and assembled the genome of O. corvina and used a method called peptide pattern recognition to identify 73 different proteases. Comparative genome analysis...... broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro....... A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed....

  2. Comparison of the genomes and transcriptomes associated with the different protease secretions of Aspergillus oryzae 100-8 and 3.042.

    Science.gov (United States)

    Zhao, Guozhong; Yao, Yunping; Hou, Lihua; Wang, Chunling; Cao, Xiaohong

    2014-10-01

    Aspergillus oryzae is used to produce traditional fermented foods and beverages. A. oryzae 3.042 produces a neutral protease and an alkaline protease but rarely an acid protease, which is unfavourable to soy-sauce fermentation. A. oryzae 100-8 was obtained by N(+) ion implantation mutagenesis of A. oryzae 3.042, and the protease secretions of these two strains are different. Sequencing the genome of A. oryzae 100-8 and comparing it to the genomes of A. oryzae 100-8 and 3.042 revealed some differences, such as single nucleotide polymorphisms, nucleotide deletion or insertion. Some of these differences may reflect the ability of A. oryzae to secrete proteases. Transcriptional sequencing and analysis of the two strains during the same growth processes provided further insights into the genes and pathways involved in protease secretion.

  3. Modulation of Immunological Pathways in Autistic and Neurotypical Lymphoblastoid Cell Lines by the Enteric Microbiome Metabolite Propionic Acid.

    Science.gov (United States)

    Frye, Richard E; Nankova, Bistra; Bhattacharyya, Sudeepa; Rose, Shannon; Bennuri, Sirish C; MacFabe, Derrick F

    2017-01-01

    Propionic acid (PPA) is a ubiquitous short-chain fatty acid which is a fermentation product of the enteric microbiome and present or added to many foods. While PPA has beneficial effects, it is also associated with human disorders, including autism spectrum disorders (ASDs). We previously demonstrated that PPA modulates mitochondrial dysfunction differentially in subsets of lymphoblastoid cell lines (LCLs) derived from patients with ASD. Specifically, PPA significantly increases mitochondrial function in LCLs that have mitochondrial dysfunction at baseline [individuals with autistic disorder with atypical mitochondrial function (AD-A) LCLs] as compared to ASD LCLs with normal mitochondrial function [individuals with autistic disorder with normal mitochondrial function (AD-N) LCLs] and control (CNT) LCLs. PPA at 1 mM was found to have a minimal effect on expression of immune genes in CNT and AD-N LCLs. However, as hypothesized, Panther analysis demonstrated that 1 mM PPA exposure at 24 or 48 h resulted in significant activation of the immune system genes in AD-A LCLs. When the effect of PPA on ASD LCLs were compared to the CNT LCLs, both ASD groups demonstrated immune pathway activation, although the AD-A LCLs demonstrate a wider activation of immune genes. Ingenuity Pathway Analysis identified several immune-related pathways as key Canonical Pathways that were differentially regulated, specifically human leukocyte antigen expression and immunoglobulin production genes were upregulated. These data demonstrate that the enteric microbiome metabolite PPA can evoke atypical immune activation in LCLs with an underlying abnormal metabolic state. As PPA, as well as enteric bacteria which produce PPA, have been implicated in a wide variety of diseases which have components of immune dysfunction, including ASD, diabetes, obesity, and inflammatory diseases, insight into this metabolic modulator may have wide applications for both health and disease.

  4. In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

    Science.gov (United States)

    Greindl, Melanie; Föger, Florian; Hombach, Juliane; Bernkop-Schnürch, Andreas

    2009-08-01

    Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA(250)-Cys(,) displaying 580 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA(250) or 4% (w/v) thiolated PAA(250) were orally given to rats. The PAA(250)-Cys solution increased the area under the plasma concentration-time curve (AUC(0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA(250). This in vivo study revealed that PAA(250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.

  5. Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice.

    Directory of Open Access Journals (Sweden)

    Alice Chaplin

    Full Text Available The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat or a high-fat (HF, 43% kJ content as fat diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkermansia muciniphila. However, CLA supplementation was not able to override the negative effects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.

  6. Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice.

    Science.gov (United States)

    Chaplin, Alice; Parra, Pilar; Serra, Francisca; Palou, Andreu

    2015-01-01

    The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkermansia muciniphila. However, CLA supplementation was not able to override the negative effects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.

  7. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  8. Nitrate uptake and utilization is modulated by exogenous gamma-aminobutyric acid in Arabidopsis thaliana seedlings.

    Science.gov (United States)

    Barbosa, Jose M; Singh, Narendra K; Cherry, Joe H; Locy, Robert D

    2010-06-01

    Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO(3)(-)) in the growth medium. At NO(3)(-) concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO(3)(-), the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO(3)(-) uptake at low NO(3)(-), while GABA inhibited NO(3)(-) uptake at high NO(3)(-). Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.

  9. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

    Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

    2002-01-01

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  10. Extracellular proteases of Trichoderma species. A review.

    Science.gov (United States)

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  11. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

    Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

  12. Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

    Science.gov (United States)

    Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

    2012-08-24

    Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

  13. Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

    Science.gov (United States)

    Taga, Yuki; Hayashida, Osamu; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2017-09-01

    Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P 2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of ginger protease can be used as a functional food for patients with type 2 diabetes.

  14. CD36 Modulates Fasting and Preabsorptive Hormone and Bile Acid Levels.

    Science.gov (United States)

    Shibao, Cyndya A; Celedonio, Jorge E; Tamboli, Robyn; Sidani, Reem; Love-Gregory, Latisha; Pietka, Terri; Xiong, Yanhua; Wei, Yan; Abumrad, Naji N; Abumrad, Nada A; Flynn, Charles Robb

    2018-05-01

    Abnormal fatty acid (FA) metabolism contributes to diabetes and cardiovascular disease. The FA receptor CD36 has been linked to risk of metabolic syndrome. In rodents CD36 regulates various aspects of fat metabolism, but whether it has similar actions in humans is unknown. We examined the impact of a coding single-nucleotide polymorphism in CD36 on postprandial hormone and bile acid (BA) responses. To examine whether the minor allele (G) of coding CD36 variant rs3211938 (G/T), which reduces CD36 level by ∼50%, influences hormonal responses to a high-fat meal (HFM). Obese African American (AA) women carriers of the G allele of rs3211938 (G/T) and weight-matched noncarriers (T/T) were studied before and after a HFM. Two-center study. Obese AA women. HFM. Early preabsorptive responses (10 minutes) and extended excursions in plasma hormones [C-peptide, insulin, incretins, ghrelin fibroblast growth factor (FGF)19, FGF21], BAs, and serum lipoproteins (chylomicrons, very-low-density lipoprotein) were determined. At fasting, G-allele carriers had significantly reduced cholesterol and glycodeoxycholic acid and consistent but nonsignificant reductions of serum lipoproteins. Levels of GLP-1 and pancreatic polypeptide (PP) were reduced 60% to 70% and those of total BAs were 1.8-fold higher. After the meal, G-allele carriers displayed attenuated early (-10 to 10 minute) responses in insulin, C-peptide, GLP-1, gastric inhibitory peptide, and PP. BAs exhibited divergent trends in G allele carriers vs noncarriers concomitant with differential FGF19 responses. CD36 plays an important role in the preabsorptive hormone and BA responses that coordinate brain and gut regulation of energy metabolism.

  15. Dietary fatty acids modulate the effects of TNF family apoptotic inducers

    Czech Academy of Sciences Publication Activity Database

    Hofmanová, Jiřina; Vaculová, Alena; Hýžďalová, Martina; Kočí, Lenka; Skender, Belma; Kozubík, Alois

    2009-01-01

    Roč. 24, č. 1 (2009), s. 353 ISSN 1107-3756. [The 14th World Congress on Advances in Oncology, and 12th International Symposium on Molecular Medicine . 15.10.2009-17.10.2009, Loutraki] R&D Projects: GA ČR(CZ) GA524/07/1178; GA AV ČR(CZ) 1QS500040507 Grant - others:GA ČR(CZ) GA305/09/1526 Program:GA Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : fatty acids * apoptosis * colon cancer Subject RIV: BO - Biophysics

  16. Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4.

    Directory of Open Access Journals (Sweden)

    Byoung-Kuk Na

    2010-10-01

    Full Text Available Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA and Z-Phe-Arg-MCA was highest at acidic pH (5.5, whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5. VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5. Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180. Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular

  17. Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

    Directory of Open Access Journals (Sweden)

    Titilayo Olufunke Femi-Ola

    2012-09-01

    Full Text Available Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis.Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that ofLactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis,Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not

  18. Fatty acid nitroalkenes induce resistance to ischemic cardiac injury by modulating mitochondrial respiration at complex II

    Directory of Open Access Journals (Sweden)

    Jeffrey R. Koenitzer

    2016-08-01

    Full Text Available Nitro-fatty acids (NO2-FA are metabolic and inflammatory-derived electrophiles that mediate pleiotropic signaling actions. It was hypothesized that NO2-FA would impact mitochondrial redox reactions to induce tissue-protective metabolic shifts in cells. Nitro-oleic acid (OA-NO2 reversibly inhibited complex II-linked respiration in isolated rat heart mitochondria in a pH-dependent manner and suppressed superoxide formation. Nitroalkylation of Fp subunit was determined by BME capture and the site of modification by OA-NO2 defined by mass spectrometric analysis. These effects translated into reduced basal and maximal respiration and favored glycolytic metabolism in H9C2 cardiomyoblasts as assessed by extracellular H+ and O2 flux analysis. The perfusion of NO2-FA induced acute cardioprotection in an isolated perfused heart ischemia/reperfusion (IR model as evidenced by significantly higher rate-pressure products. Together these findings indicate that NO2-FA can promote cardioprotection by inducing a shift from respiration to glycolysis and suppressing reactive species formation in the post-ischemic interval.

  19. Dynamic modulation of platelet aggregation, albumin and nonesterified fatty acids during physical exercise in Thoroughbred horses.

    Science.gov (United States)

    Arfuso, F; Giannetto, C; Giudice, E; Fazio, F; Piccione, G

    2016-02-01

    The effect of exercise on platelet aggregation, albumin and nonesterified fatty acids (NEFAs) values and the correlation among these parameters were evaluated in ten clinically healthy and regularly trained Thoroughbred horses. All horses were subjected to two simulated races. Blood samples were collected by jugular venipuncture before and after the first simulated race (T0PRE and T0POST), every 7 days at rest condition for a month (T1R-T2R-T3R), and before and after the second simulated race (T4PRE and T4POST) in order to assess platelet aggregation, albumin and nonesterified fatty acids (NEFAs) levels. One-way analysis of variance showed a significant effect of exercise (P<0.01) on platelet aggregation, albumin and NEFAs values. A negative correlation between platelet aggregation and albumin or NEFAs values, and a positive correlation between albumin and NEFAs values, were found both at T0POST and T4POST (P<0.05). These findings are likely related to dynamic physiological adaptations to exercise that allow re-establishment of the homeostatic equilibrium of the organism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

    Directory of Open Access Journals (Sweden)

    Frans Kurnia

    2018-02-01

    Full Text Available Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

  1. Subtype-specific Modulation of Acid-sensing Ion Channel (ASIC) Function by 2-Guanidine-4-methylquinazoline*

    Science.gov (United States)

    Alijevic, Omar; Kellenberger, Stephan

    2012-01-01

    Acid-sensing ion channels (ASICs) are neuronal Na+-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mm, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH. PMID:22948146

  2. Subtype-specific modulation of acid-sensing ion channel (ASIC) function by 2-guanidine-4-methylquinazoline.

    Science.gov (United States)

    Alijevic, Omar; Kellenberger, Stephan

    2012-10-19

    Acid-sensing ion channels (ASICs) are neuronal Na(+)-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mM, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH.

  3. Trichoderma inoculation augments grain amino acids and mineral nutrients by modulating arsenic speciation and accumulation in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Tripathi, Pratibha; Singh, Poonam C; Mishra, Aradhana; Tripathi, Rudra D; Nautiyal, Chandra S

    2015-07-01

    Trichoderma reesei is an industrially important fungi which also imparts stress tolerance and plant growth promotion in various crops. Arsenic (As) contamination of field soils is one of the challenging problems in agriculture, posing potential threats for both human health and the environment. Plants in association with microbes are a liable method to improve metal tolerance and enhance crop productivity. Chickpea (Cicer arietinum L.), is an important grain legume providing cheap source of protein in semi-arid regions including As affected areas. In this study we report the role of T. reesei NBRI 0716 (NBRI 0716) in supporting chickpea growth and improving soil quality in As simulated conditions. NBRI 0716 modulated the As speciation and its availability to improve grain yield and quality (amino acids and mineral content) in chickpea (C. arietinum L.) plants grown in As spiked soil (100 mg As kg(-1) soil). Arsenic accumulation and speciation results indicate that arsenate [As(V)] was the dominant species in chickpea seeds and rhizosphere soil. The Trichoderma reduced total grain inorganic As (Asi) by 66% and enhanced dimethylarsonic acid (DMA) and monomethylarsinic acid (MMA) content of seed and rhizosphere soil. The results indicate a probable role of NBRI 0716 in As methylation as the possible mechanism for maneuvering As stress in chickpea. Analysis of functional diversity using carbon source utilization (Biolog) showed significant difference in diversity and evenness indices among the soil microbial rhizosphere communities. Microbial diversity loss caused by As were prevented in the presence of Trichoderma NBRI 0716. Copyright © 2015. Published by Elsevier Inc.

  4. Aerobic Exercise Modulates the Free Fatty Acids and Inflammatory Response During Obesity and Cancer Cachexia.

    Science.gov (United States)

    Teixeira, Alexandre Abilio de Souza; Lira, Fábio Santos; Pimentel, Gustavo D; Oliveira de Souza, Camila; Batatinha, Helena; Biondo, Luana A; Yamashita, Alex S; Junior, Edson A Lima; Neto, José Cesar Rosa

    2016-01-01

    White adipose tissue (WAT) is no longer considered a tissue whose main function is the storage of TAG. Since the discovery of leptin in 1994, several studies have elucidated the important role of WAT as an endocrine organ, the source of the adipokines. The low-grade inflammation observed in obese and cancer cachexia patients is explained, at least partially, by the exacerbated release of proinflammatory adipokines. Despite of the recent progress in the characterization of the various adipokines and lipokines produced by WAT, little is known about the mechanisms regulating the secretion of these molecules in different physiological and pathological circumstances. Chronic exercise is a nonpharmacological therapy employed in several chronic diseases and shows an anti-inflammatory effect through the regulation of the cytokine network. In this review, we address the potential mechanisms by which the aerobic physical exercise modulate the production and release of inflammatory adipokines, as well as the inflammation-lipolysis axis in WAT, with special focus in the therapeutic role of exercise in obesity-associated insulin resistance and cancer cachexia.

  5. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

    Science.gov (United States)

    Creelman, R A; Tierney, M L; Mullet, J E

    1992-06-01

    Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

  6. The gut microbiota modulates host amino acid and glutathione metabolism in mice

    DEFF Research Database (Denmark)

    Mardinoglu, Adil; Shoaie, Saeed; Bergentall, Mattias

    2015-01-01

    , liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV-R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism...... conventionally raised (CONV-R) and germ-free (GF) mice using gene expression data and tissue-specific genome-scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue-specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon....... To validate our predictions, we measured the level of AAs and N-acetylated AAs in the hepatic portal vein of CONV-R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV-R and GF mice accounting for the content of the diet and relative gene expression differences...

  7. Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

    Science.gov (United States)

    Saibi, Walid; Zouari, Nabil; Masmoudi, Khaled; Brini, Faiçal

    2016-04-01

    Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

    Directory of Open Access Journals (Sweden)

    Ileana Corvo

    2018-04-01

    Full Text Available Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature. Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

  9. L-CARNITINE-INDUCED MODULATION OF PLASMA FATTY ACIDS METABOLISM IN HYPERLIPIDEMIC RABBITS

    Directory of Open Access Journals (Sweden)

    Frank Hernández Rosales PhD

    2006-02-01

    mejoramiento del metabolismo de las lipoproteínas. ABSTRACTThe present study was designed to examine whether the hipocholesterolemic effect of L-carnitine supplementation is related with lipoprotein fatty acid metabolism. Fatty acid compositional and cholesterol content changes were measured in lipoproteins of six different groups of rabbits. Group 1, rabbits fed a standard diet; group 2, rabbits fed standard diet plus L-carnitine 80 mg/kg bw; group 3, rabbits fed a 0.5 % cholesterol diet; group 4, rabbits fed a 0.5 % cholesterol diet plus L-carnitine 80 mg/kg b.w. These four groups were fed their diets during 126 days. Group 5 and 6 were fed the same diet as group 4 in a previous period of 126 days, and after this time, group 5 was fed the same diet as group 1, and group 6 fed the same diet as group 2, during a second period of 65 days.However, the progression of hypercholesterolemia was reduced 50 % by L-carnitine administration in those animals fed cholesterol diet. Fatty acid compositional changes in lipoprotein-cholesteryl esters were found in all groups of animals supplemented with L-carnitine. During the standard-fed period the saturated and unsaturated fatty acid ratio was increased in VLDL and HDL particles whereas was decreased in LDL. In the hyperlipidemia progression period the saturated to unsaturated fatty acid ratio in HDL fraction was slightly enhanced and in the VLDL+LDL modified particle was diminished. In the hyperlipidemia regression period, plasma cholesterol level was additionally reduced in a 33 % in the group 6; and the saturated to unsaturated fatty ratio had the same behaviour from that observed in the progression period for HDL and VLDL+LDL particles. A remarkable reduction (75% of aorta atherosclerotic plaques in the group 6 was found. From these results we concluded that L-carnitine, in this experimental model, induces an improved lipoprotein metabolism.

  10. Endogenous salicylic acid is required for promoting cadmium tolerance of Arabidopsis by modulating glutathione metabolisms

    International Nuclear Information System (INIS)

    Guo, Bin; Liu, Chen; Li, Hua; Yi, Keke; Ding, Nengfei; Li, Ningyu; Lin, Yicheng; Fu, Qinglin

    2016-01-01

    Highlights: • The role of endogenous SA in mediating Cd tolerance was explored using sid2 mutants. • Cd stress induces SA accumulation in a SID2 dependent way. • Depletion of SA causes negative effects on Cd tolerance. • Endogenous SA is required for promoting Cd tolerance by modulating GSH metabolism. • Possible mode of SA signaling through GR/GSH pathway under Cd toxicity was discussed. - Abstract: A few studies with NahG transgenic lines of Arabidopsis show that depletion of SA enhances cadmium (Cd) tolerance. However, it remains some uncertainties that the defence signaling may be a result of catechol accumulation in NahG transgenic lines but not SA deficiency. Here, we conducted a set of hydroponic assays with another SA-deficient mutant sid2 to examine the endogenous roles of SA in Cd tolerance, especially focusing on the glutathione (GSH) cycling. Our results showed that reduced SA resulted in negative effects on Cd tolerance, including decreased Fe uptake and chlorophyll concentration, aggravation of oxidative damage and growth inhibition. Cd exposure significantly increased SA concentration in wild-type leaves, but did not affect it in sid2 mutants. Depletion of SA did not disturb the Cd uptake in either roots or shoots. The reduced Cd tolerance in sid2 mutants is due to the lowered GSH status, which is associated with the decreased expression of serine acetyltransferase along with a decline in contents of non-protein thiols, phytochelatins, and the lowered transcription and activities of glutathione reductase1 (GR1) which reduced GSH regeneration. Finally, the possible mode of SA signaling through the GR/GSH pathway during Cd exposure is discussed.

  11. Endogenous salicylic acid is required for promoting cadmium tolerance of Arabidopsis by modulating glutathione metabolisms

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Bin, E-mail: ndgb@163.com [Institute of Environment, Resource, Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences, Geological Research Center For Agricultural Applications, China Geological Survey, Hangzhou (China); Liu, Chen; Li, Hua [Institute of Environment, Resource, Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences, Geological Research Center For Agricultural Applications, China Geological Survey, Hangzhou (China); Yi, Keke [Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou (China); Ding, Nengfei; Li, Ningyu; Lin, Yicheng [Institute of Environment, Resource, Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences, Geological Research Center For Agricultural Applications, China Geological Survey, Hangzhou (China); Fu, Qinglin, E-mail: fuql161@yahoo.com.cn [Institute of Environment, Resource, Soil and Fertilizer, Zhejiang Academy of Agricultural Sciences, Geological Research Center For Agricultural Applications, China Geological Survey, Hangzhou (China)

    2016-10-05

    Highlights: • The role of endogenous SA in mediating Cd tolerance was explored using sid2 mutants. • Cd stress induces SA accumulation in a SID2 dependent way. • Depletion of SA causes negative effects on Cd tolerance. • Endogenous SA is required for promoting Cd tolerance by modulating GSH metabolism. • Possible mode of SA signaling through GR/GSH pathway under Cd toxicity was discussed. - Abstract: A few studies with NahG transgenic lines of Arabidopsis show that depletion of SA enhances cadmium (Cd) tolerance. However, it remains some uncertainties that the defence signaling may be a result of catechol accumulation in NahG transgenic lines but not SA deficiency. Here, we conducted a set of hydroponic assays with another SA-deficient mutant sid2 to examine the endogenous roles of SA in Cd tolerance, especially focusing on the glutathione (GSH) cycling. Our results showed that reduced SA resulted in negative effects on Cd tolerance, including decreased Fe uptake and chlorophyll concentration, aggravation of oxidative damage and growth inhibition. Cd exposure significantly increased SA concentration in wild-type leaves, but did not affect it in sid2 mutants. Depletion of SA did not disturb the Cd uptake in either roots or shoots. The reduced Cd tolerance in sid2 mutants is due to the lowered GSH status, which is associated with the decreased expression of serine acetyltransferase along with a decline in contents of non-protein thiols, phytochelatins, and the lowered transcription and activities of glutathione reductase1 (GR1) which reduced GSH regeneration. Finally, the possible mode of SA signaling through the GR/GSH pathway during Cd exposure is discussed.

  12. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimina......Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...... for ADAM 12 involving both furin cleavage and copper binding....

  13. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

  14. Identification of key amino acid residues modulating intracellular and in vitro microcin E492 amyloid formation

    Directory of Open Access Journals (Sweden)

    Paulina eAguilera

    2016-01-01

    Full Text Available Microcin E492 (MccE492 is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well characterized, however it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in E. coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophillic probes, 2-4´-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59, which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  15. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium.

    Science.gov (United States)

    Santos, Anderson F; Valle, Roberta S; Pacheco, Clarissa A; Alvarez, Vanessa M; Seldin, Lucy; Santos, André L S

    2013-12-01

    Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

  16. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

  17. Food deprivation modulates gamma-aminobutyric acid receptors and peripheral benzodiazepine binding sites in rats.

    Science.gov (United States)

    Weizman, A; Bidder, M; Fares, F; Gavish, M

    1990-12-03

    The effect of 5 days of food deprivation followed by 5 days of refeeding on gamma-aminobutyric acid (GABA) receptors, central benzodiazepine receptors (CBR), and peripheral benzodiazepine binding sites (PBzS) was studied in female Sprague-Dawley rats. Starvation induced a decrease in the density of PBzS in peripheral organs: adrenal (35%; P less than 0.001), kidney (33%; P less than 0.01), and heart (34%; P less than 0.001). Restoration of [3H]PK 11195 binding to normal values was observed in all three organs after 5 days of refeeding. The density of PBzS in the ovary, pituitary, and hypothalamus was not affected by starvation. Food deprivation resulted in a 35% decrease in cerebellar GABA receptors (P less than 0.01), while CBR in the hypothalamus and cerebral cortex remained unaltered. The changes in PBzS observed in the heart and kidney may be related to the long-term metabolic stress associated with starvation and to the functional changes occurring in these organs. The down-regulation of the adrenal PBzS is attributable to the suppressive effect of hypercortisolemia on pituitary ACTH release. The reduction in cerebellar GABA receptors may be an adaptive response to food deprivation stress and may be relevant to the proaggressive effect of hunger.

  18. Modulation of Tamoxifen Cytotoxicity by Caffeic Acid Phenethyl Ester in MCF-7 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tarek K. Motawi

    2016-01-01

    Full Text Available Although Tamoxifen (TAM is one of the most widely used drugs in managing breast cancer, many women still relapse after long-term therapy. Caffeic acid phenethyl ester (CAPE is a polyphenolic compound present in many medicinal plants and in propolis. The present study examined the effect of CAPE on TAM cytotoxicity in MCF-7 cells. MCF-7 cells were treated with different concentrations of TAM and/or CAPE for 48 h. This novel combination exerted synergistic cytotoxic effects against MCF-7 cells via induction of apoptotic machinery with activation of caspases and DNA fragmentation, along with downregulation of Bcl-2 and Beclin 1 expression levels. However, the mammalian microtubule-associated protein light chain LC 3-II level was unchanged. Vascular endothelial growth factor level was also decreased, whereas levels of glutathione and nitric oxide were increased. In conclusion, CAPE augmented TAM cytotoxicity via multiple mechanisms, providing a novel therapeutic approach for breast cancer treatment that can overcome resistance and lower toxicity. This effect provides a rationale for further investigation of this combination.

  19. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jason R Gerstner

    2011-01-01

    Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

  20. Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease.

    Science.gov (United States)

    Lee, Jong Kil; Jin, Hee Kyung; Park, Min Hee; Kim, Bo-ra; Lee, Phil Hyu; Nakauchi, Hiromitsu; Carter, Janet E; He, Xingxuan; Schuchman, Edward H; Bae, Jae-sung

    2014-07-28

    In Alzheimer's disease (AD), abnormal sphingolipid metabolism has been reported, although the pathogenic consequences of these changes have not been fully characterized. We show that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, leading to defective autophagic degradation due to lysosomal depletion. Partial genetic inhibition of ASM (ASM(+/-)) in a mouse model of familial AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autophagocytic defect by restoring lysosomal biogenesis, resulting in improved AD clinical and pathological findings, including reduction of amyloid-β (Aβ) deposition and improvement of memory impairment. Similar effects were noted after pharmacologic restoration of ASM to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition. Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM inhibition is a potential new therapeutic intervention for the disease. © 2014 Lee et al.

  1. Fatty acid modulated human serum albumin binding of the β-carboline alkaloids norharmane and harmane.

    Science.gov (United States)

    Domonkos, Celesztina; Fitos, Ilona; Visy, Júlia; Zsila, Ferenc

    2013-12-02

    Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.

  2. Short-Chain Fatty Acids Enhance the Lipid Accumulation of 3T3-L1 Cells by Modulating the Expression of Enzymes of Fatty Acid Metabolism.

    Science.gov (United States)

    Yu, Haining; Li, Ran; Huang, Haiyong; Yao, Ru; Shen, Shengrong

    2018-01-01

    Short-chain fatty acids (SCFA) such as acetic acid, propionic acid, and butyric acid are produced by fermentation by gut microbiota. In this paper, we investigate the effects of SCFA on 3T3-L1 cells and the underlying molecular mechanisms. The cells were treated with acetic acid, propionic acid, or butyric acid when cells were induced to differentiate into adipocytes. MTT assay was employed to detect the viability of 3T3-L1 cells. Oil Red O staining was used to visualize the lipid content in 3T3-L1 cells. A triglyceride assay kit was used to detect the triacylglycerol content in 3T3-L1 cells. qRT-PCR and Western blot were used to evaluate the expression of metabolic enzymes. MTT results showed that safe concentrations of acetic acid, propionic acid, and butyric acid were less than 6.4, 3.2, and 0.8 mM, respectively. Oil Red O staining and triacylglycerols detection results showed that treatment with acetic acid, propionic acid, and butyric acid accelerated the 3T3-L1 adipocyte differentiation. qRT-PCR and Western blot results showed that the expressions of lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4), fatty acid transporter protein 4 (FATP4), and fatty acid synthase (FAS) were significantly increased by acetic acid, propionic acid, and butyric acid treatment during adipose differentiation (p fatty acid metabolism. © 2018 AOCS.

  3. Effects of gamma-aminobutyric acid-modulating drugs on working memory and brain function in patients with schizophrenia.

    Science.gov (United States)

    Menzies, Lara; Ooi, Cinly; Kamath, Shri; Suckling, John; McKenna, Peter; Fletcher, Paul; Bullmore, Ed; Stephenson, Caroline

    2007-02-01

    Cognitive impairment causes morbidity in schizophrenia and could be due to abnormalities of cortical interneurons using the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). To test the predictions that cognitive and brain functional responses to GABA-modulating drugs are correlated and abnormal in schizophrenia. Pharmacological functional magnetic resonance imaging study of 2 groups, each undergoing scanning 3 times, using an N-back working memory task, after placebo, lorazepam, or flumazenil administration. Eleven patients with chronic schizophrenia were recruited from a rehabilitation service, and 11 healthy volunteers matched for age, sex, and premorbid IQ were recruited from the local community. Intervention Participants received 2 mg of oral lorazepam, a 0.9-mg intravenous flumazenil bolus followed by a flumazenil infusion of 0.0102 mg/min, or oral and intravenous placebo. Working memory performance was summarized by the target discrimination index at several levels of difficulty. Increasing (or decreasing) brain functional activation in response to increasing task difficulty was summarized by the positive (or negative) load response. Lorazepam impaired performance and flumazenil enhanced it; these cognitive effects were more salient in schizophrenic patients. Functional magnetic resonance imaging demonstrated positive load response in a frontoparietal system and negative load response in the temporal and posterior cingulate regions; activation of the frontoparietal cortex was positively correlated with deactivation of the temporocingulate cortex. After placebo administration, schizophrenic patients had abnormally attenuated activation of the frontoparietal cortex and deactivation of the temporocingulate cortex; this pattern was mimicked in healthy volunteers and exacerbated in schizophrenic patients by lorazepam. However, in schizophrenic patients, flumazenil enhanced deactivation of the temporocingulate and activation of the anterior cingulate

  4. Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling.

    Science.gov (United States)

    Futrega, Kathryn; Yu, Jianshi; Jones, Jace W; Kane, Maureen A; Lott, William B; Atkinson, Kerry; Doran, Michael R

    2016-04-21

    Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.

  5. PPARalpha-dependent modulation of hepatic CYP1A by clofibric acid in rats.

    Science.gov (United States)

    Shaban, Zein; El-Shazly, Samir; Ishizuka, Mayumi; Kimura, Kazuhiro; Kazusaka, Akio; Fujita, Shoichi

    2004-09-01

    Fibrates, hypolipidemic drugs, have been reported to suppress the metabolic activities of cytochrome P450 1A1 and 1A2 in rats but the mechanism has not been elucidated. In the present study we tested the hypothesis that the inhibitory effect of fibrates on arylhydrocarbon receptor (AhR) function may be due to their stimulatory effects on PPARalpha. Sudan III (S.III) treatment induced CYP 1A1 and CYP 1A2 protein expression, mRNA and their metabolic activities, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD), in Wistar rats higher than those in the control. Co-treatment of rats with S.III and clofibric acid (CA) caused a 40-50% decrease in the induced levels of CYP1A1 and CYP1A2 protein, mRNA expression and their metabolic activities and reduced AhR protein expression. When we treated HepG2 cells with S.III and/or CA, no suppressive effect on S.III-induced CYP1A1 protein expression due to CA was found. HepG2 cells were transiently transfected with increasing concentrations of PPARalpha mammalian expression vector and exposed to the same treatment. CA co-treatment with S.III decreased AhR protein and S.III-induced CYP1A1 protein expression with increasing dose of PPARalpha transfected into HepG2 cells. Our results demonstrate that the suppressive effect of fibrates on CYP1A is PPARalpha-dependent and suggest that PPARalpha has an inhibitory effect on AhR function.

  6. Long-Chain Omega-3 Polyunsaturated Fatty Acids Modulate Mammary Gland Composition and Inflammation.

    Science.gov (United States)

    Khadge, Saraswoti; Thiele, Geoffrey M; Sharp, John Graham; McGuire, Timothy R; Klassen, Lynell W; Black, Paul N; DiRusso, Concetta C; Talmadge, James E

    2018-03-25

    Studies in rodents have shown that dietary modifications as mammary glands (MG) develop, regulates susceptibility to mammary tumor initiation. However, the effects of dietary PUFA composition on MGs in adult life, remains poorly understood. This study investigated morphological alterations and inflammatory microenvironments in the MGs of adult mice fed isocaloric and isolipidic liquid diets with varying compositions of omega (ω)-6 and long-chain (Lc)-ω3FA that were pair-fed. Despite similar consumption levels of the diets, mice fed the ω-3 diet had significantly lower body-weight gains, and abdominal-fat and mammary fat pad (MFP) weights. Fatty acid analysis showed significantly higher levels of Lc-ω-3FAs in the MFPs of mice on the ω-3 diet, while in the MFPs from the ω-6 group, Lc-ω-3FAs were undetectable. Our study revealed that MGs from ω-3 group had a significantly lower ductal end-point density, branching density, an absence of ductal sprouts, a thinner ductal stroma, fewer proliferating epithelial cells and a lower transcription levels of estrogen receptor 1 and amphiregulin. An analysis of the MFP and abdominal-fat showed significantly smaller adipocytes in the ω-3 group, which was accompanied by lower transcription levels of leptin, IGF1, and IGF1R. Further, MFPs from the ω-3 group had significantly decreased numbers and sizes of crown-like-structures (CLS), F4/80+ macrophages and decreased expression of proinflammatory mediators including Ptgs2, IL6, CCL2, TNFα, NFκB, and IFNγ. Together, these results support dietary Lc-ω-3FA regulation of MG structure and density and adipose tissue inflammation with the potential for dietary Lc-ω-3FA to decrease the risk of mammary gland tumor formation.

  7. Bile acids modulate signaling by functional perturbation of plasma membrane domains.

    Science.gov (United States)

    Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

    2013-12-13

    Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

  8. The Rheological Properties of Lipid Monolayers Modulate the Incorporation of l-Ascorbic Acid Alkyl Esters.

    Science.gov (United States)

    Díaz, Yenisleidy de Las Mercedes Zulueta; Mottola, Milagro; Vico, Raquel V; Wilke, Natalia; Fanani, María Laura

    2016-01-19

    In this work, we tested the hypothesis that the incorporation of amphiphilic drugs into lipid membranes may be regulated by their rheological properties. For this purpose, two members of the l-ascorbic acid alkyl esters family (ASCn) were selected, ASC16 and ASC14, which have different rheological properties when organized at the air/water interface. They are lipophilic forms of vitamin C used in topical pharmacological preparations. The effect of the phase state of the host lipid membranes on ASCn incorporation was explored using Langmuir monolayers. Films of pure lipids with known phase states have been selected, showing liquid-expanded, liquid-condensed, and solid phases as well as pure cholesterol films in liquid-ordered state. We also tested ternary and quaternary mixed films that mimic the properties of cholesterol containing membranes and of the stratum corneum. The compressibility and shear properties of those monolayers were assessed in order to define its phase character. We found that the length of the acyl chain of the ASCn compounds induces differential changes in the rheological properties of the host membrane and subtly regulates the kinetics and extent of the penetration process. The capacity for ASCn uptake was found to depend on the phase state of the host film. The increase in surface pressure resultant after amphiphile incorporation appears to be a function of the capacity of the host membrane to incorporate such amphiphile as well as the rheological response of the film. Hence, monolayers that show a solid phase state responded with a larger surface pressure increase to the incorporation of a comparable amount of amphiphile than liquid-expanded ones. The cholesterol-containing films, including the mixture that mimics stratum corneum, allowed a very scarce ASCn uptake independently of the membrane diffusional properties. This suggests an important contribution of Cho on the maintenance of the barrier function of stratum corneum.

  9. Feeding of the water extract from Ganoderma lingzhi to rats modulates secondary bile acids, intestinal microflora, mucins, and propionate important to colon cancer.

    Science.gov (United States)

    Yang, Yongshou; Nirmagustina, Dwi Eva; Kumrungsee, Thanutchaporn; Okazaki, Yukako; Tomotake, Hiroyuki; Kato, Norihisa

    2017-09-01

    Consumption of reishi mushroom has been reported to prevent colon carcinogenesis in rodents, although the underlying mechanisms remain unclear. To investigate this effect, rats were fed a high-fat diet supplemented with 5% water extract from either the reishi mushroom (Ganoderma lingzhi) (WGL) or the auto-digested reishi G. lingzhi (AWGL) for three weeks. Both extracts markedly reduced fecal secondary bile acids, such as lithocholic acid and deoxycholic acid (colon carcinogens). These extracts reduced the numbers of Clostridium coccoides and Clostridium leptum (secondary bile acids-producing bacteria) in a per g of cecal digesta. Fecal mucins and cecal propionate were significantly elevated by both extracts, and fecal IgA was significantly elevated by WGL, but not by AWGL. These results suggest that the reishi extracts have an impact on colon luminal health by modulating secondary bile acids, microflora, mucins, and propionate that related to colon cancer.

  10. In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation

    International Nuclear Information System (INIS)

    Fernández-Moriano, Carlos; Divakar, Pradeep Kumar; Crespo, Ana; Gómez-Serranillos, M. Pilar

    2017-01-01

    The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H 2 O 2 was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines). We found that a pre-treatment with FUM significantly enhanced cell viability compared to H 2 O 2 -treated cells, and we selected the optimal concentrations in each model (1 and 25 μg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H 2 O 2 . It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H 2 O 2 -induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders. - Highlights: • FUM pre-treatment exerts significant cytoprotection against H 2 O 2 -mediated apoptosis. • ROS

  11. In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Moriano, Carlos [Department of Pharmacology, Faculty of Pharmacy, University Complutense of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid (Spain); Divakar, Pradeep Kumar; Crespo, Ana [Department of Plant Biology II, Faculty of Pharmacy, University Complutense of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid (Spain); Gómez-Serranillos, M. Pilar, E-mail: pserra@ucm.es [Department of Pharmacology, Faculty of Pharmacy, University Complutense of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid (Spain)

    2017-02-01

    The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H{sub 2}O{sub 2} was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines). We found that a pre-treatment with FUM significantly enhanced cell viability compared to H{sub 2}O{sub 2}-treated cells, and we selected the optimal concentrations in each model (1 and 25 μg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H{sub 2}O{sub 2}. It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H{sub 2}O{sub 2}-induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders. - Highlights: • FUM pre-treatment exerts significant cytoprotection against H

  12. Modulation of fibroblast growth factor 19 expression by bile acids, meal replacement and energy drinks, milk, and coffee.

    Directory of Open Access Journals (Sweden)

    Amanda M Styer

    Full Text Available BACKGROUND: The enterohepatic pathway involving the fibroblast growth factor 19 (FGF19 and bile acids (BA has been linked with the etiology and remission of type 2 diabetes (T2D following Roux-en-Y gastric bypass (RYGB surgery. Specifically, diabetic patients had lower FGF19 circulating levels but postoperative FGF19 and BA levels were higher in diabetic patients that experience remission of T2D, as compared to non-diabetic patients and diabetic patients that do not experience remission. It has been proposed that this may be due to the direct flow of digestate-free bile acids into the ileum benefiting mostly T2D patients without severe diabetes. METHODS/RESULTS: We used a human colorectal cell line (LS174T that endogenously expresses FGF19, real time PCR, and Elisas for precise quantitation of FGF19 mRNA and secreted protein levels. We report here that BA and fractions of BA stimulated FGF19 in vitro but this effect was partially blocked when BA were pre-incubated with a lipoprotein mix which emulates digested food. In addition, we show that FGF19 mRNA was stimulated by meal replacement drinks (Ensure, Glucerna, SlimFast, non-fat milk, and coffee which has been linked with reduced risk for developing diabetes. Pure caffeine and the 5-hour Energy drink, on the other hand, decreased FGF19 mRNA. CONCLUSIONS: In summary, FGF19 expression in vitro is modifiable by popular drinks suggesting that such approaches could potentially be used for modulating FGF19 expression in humans.

  13. Modulation of fibroblast growth factor 19 expression by bile acids, meal replacement and energy drinks, milk, and coffee.

    Science.gov (United States)

    Styer, Amanda M; Roesch, Stephen L; Argyropoulos, George

    2014-01-01

    The enterohepatic pathway involving the fibroblast growth factor 19 (FGF19) and bile acids (BA) has been linked with the etiology and remission of type 2 diabetes (T2D) following Roux-en-Y gastric bypass (RYGB) surgery. Specifically, diabetic patients had lower FGF19 circulating levels but postoperative FGF19 and BA levels were higher in diabetic patients that experience remission of T2D, as compared to non-diabetic patients and diabetic patients that do not experience remission. It has been proposed that this may be due to the direct flow of digestate-free bile acids into the ileum benefiting mostly T2D patients without severe diabetes. We used a human colorectal cell line (LS174T) that endogenously expresses FGF19, real time PCR, and Elisas for precise quantitation of FGF19 mRNA and secreted protein levels. We report here that BA and fractions of BA stimulated FGF19 in vitro but this effect was partially blocked when BA were pre-incubated with a lipoprotein mix which emulates digested food. In addition, we show that FGF19 mRNA was stimulated by meal replacement drinks (Ensure, Glucerna, SlimFast), non-fat milk, and coffee which has been linked with reduced risk for developing diabetes. Pure caffeine and the 5-hour Energy drink, on the other hand, decreased FGF19 mRNA. In summary, FGF19 expression in vitro is modifiable by popular drinks suggesting that such approaches could potentially be used for modulating FGF19 expression in humans.

  14. gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells

    International Nuclear Information System (INIS)

    Gallo, V.; Wise, B.C.; Vaccarino, F.; Guidotti, A.

    1985-01-01

    t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, the authors have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [ 35 S]TBPS. At 25 degrees C, and in a modified Locke solution, the [ 35 S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [ 35 S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [ 35 S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [ 35 S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [ 35 S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [ 35 S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam

  15. An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition

    Directory of Open Access Journals (Sweden)

    Gardiner Kristin L

    2011-10-01

    Full Text Available Abstract Background The evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents. Methods One fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D under varying conditions and the outcome of competition measured over 14 days by flow cytometry. Results We demonstrate that ascorbic acid (vitamin C can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus. Conclusions This model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.

  16. Dietary fatty acids modulate associations between genetic variants and circulating fatty acids in plasma and erythrocyte membranes: meta-analysis of 9 studies in the CHARGE consortium

    Science.gov (United States)

    Smith, Caren E.; Follis, Jack L.; Nettleton, Jennifer A.; Foy, Millennia; Wu, Jason H.Y.; Ma, Yiyi; Tanaka, Toshiko; Manichakul, Ani W.; Wu, Hongyu; Chu, Audrey Y.; Steffen, Lyn M.; Fornage, Myriam; Mozaffarian, Dariush; Kabagambe, Edmond K.; Ferruci, Luigi; da Chen, Yii-Der I; Rich, Stephen S.; Djoussé, Luc; Ridker, Paul M.; Tang, Weihong; McKnight, Barbara; Tsai, Michael Y.; Bandinelli, Stefania; Rotter, Jerome I.; Hu, Frank B.; Chasman, Daniel I.; Psaty, Bruce M.; Arnett, Donna K.; King, Irena B.; Sun, Qi; Wang, Lu; Lumley, Thomas; Chiuve, Stephanie E.; Siscovick, David S; Ordovás, José M.; Lemaitre, Rozenn N.

    2015-01-01

    Scope Tissue concentrations of omega-3 fatty acids may reduce cardiovascular disease risk, and genetic variants are associated with circulating fatty acids concentrations. Whether dietary fatty acids interact with genetic variants to modify circulating omega-3 fatty acids is unclear. Objective We evaluated interactions between genetic variants and fatty acid intakes for circulating alpha-linoleic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). Methods and Results We conducted meta-analyses (N to 11,668) evaluating interactions between dietary fatty acids and genetic variants (rs174538 and rs174548 in FADS1 (fatty acid desaturase 1), rs7435 in AGPAT3 (1-acyl-sn-glycerol-3-phosphate), rs4985167 in PDXDC1 (pyridoxal-dependent decarboxylase domain-containing 1), rs780094 in GCKR (glucokinase regulatory protein) and rs3734398 in ELOVL2 (fatty acid elongase 2)). Stratification by measurement compartment (plasma vs. erthyrocyte) revealed compartment-specific interactions between FADS1 rs174538 and rs174548 and dietary ALA and linoleic acid for DHA and DPA. Conclusion Our findings reinforce earlier reports that genetically-based differences in circulating fatty acids may be partially due to differences in the conversion of fatty acid precursors. Further, fatty acids measurement compartment may modify gene-diet relationships, and considering compartment may improve the detection of gene-fatty acids interactions for circulating fatty acid outcomes. PMID:25626431

  17. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

    Science.gov (United States)

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

    2011-02-11

    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  18. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    International Nuclear Information System (INIS)

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-01-01

    Highlights: → Novel role for poliovirus 2A protease as splicing modulator. → Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. → Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A pro modulating the alternative splicing of pre-mRNAs. Expression of 2A pro potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A pro abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A pro , leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A pro on splicing is to selectively block the second catalytic step.

  19. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  20. Discovery and Early Clinical Evaluation of BMS-605339, a Potent and Orally Efficacious Tripeptidic Acylsulfonamide NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

    Energy Technology Data Exchange (ETDEWEB)

    Scola, Paul M.; Wang, Alan Xiangdong; Good, Andrew C.; Sun, Li-Qiang; Combrink, Keith D.; Campbell, Jeffrey A.; Chen, Jie; Tu, Yong; Sin, Ny; Venables, Brian L.; Sit, Sing-Yuen; Chen, Yan; Cocuzza, Anthony; Bilder, Donna M.; D’Andrea, Stanley; Zheng, Barbara; Hewawasam, Piyasena; Ding, Min; Thuring, Jan; Li, Jianqing; Hernandez, Dennis; Yu, Fei; Falk, Paul; Zhai, Guangzhi; Sheaffer, Amy K.; Chen, Chaoqun; Lee, Min S.; Barry, Diana; Knipe, Jay O.; Li, Wenying; Han, Yong-Hae; Jenkins, Susan; Gesenberg, Christoph; Gao, Qi; Sinz, Michael W.; Santone, Kenneth S.; Zvyaga, Tatyana; Rajamani, Ramkumar; Klei, Herbert E.; Colonno, Richard J.; Grasela, Dennis M.; Hughes, Eric; Chien, Caly; Adams, Stephen; Levesque, Paul C.; Li, Danshi; Zhu, Jialong; Meanwell, Nicholas A.; McPhee, Fiona

    2014-03-13

    The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

  1. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

    2013-01-01

    abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

  2. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    Science.gov (United States)

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  3. Omega-3 Fatty Acids Supplementation Differentially Modulates the SDF-1/CXCR-4 Cell Homing Axis in Hypertensive and Normotensive Rats.

    Science.gov (United States)

    Halmenschlager, Luiza; Lehnen, Alexandre Machado; Marcadenti, Aline; Markoski, Melissa Medeiros

    2017-08-01

    We assessed the effect of acute and chronic dietary supplementation of ω-3 on lipid metabolism and cardiac regeneration, through its influence on the Stromal Derived Factor-1 (SDF-1) and its receptor (CXCR4) axis in normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were allocated in eight groups (of eight animals each), which received daily orogastric administration of ω-3 (1 g) for 24 h, 72 h or 2 weeks. Blood samples were collected for the analysis of the lipid profile and SDF-1 systemic levels (ELISA). At the end of the treatment period, cardiac tissue was collected for CXCR4 expression analysis (Western blot). The use of ω-3 caused a reduction in total cholesterol levels ( p = 0.044), and acutely activated the SDF-1/CXCR4 axis in normotensive animals ( p = 0.037). In the presence of the ω-3, after 72 h, SDF-1 levels decreased in WKY and increased in SHR ( p = 0.017), and tissue expression of the receptor CXCR4 was higher in WKY than in SHR ( p = 0.001). The ω-3 fatty acid supplementation differentially modulates cell homing mediators in normotensive and hypertensive animals. While WKY rats respond acutely to omega-3 supplementation, showing increased release of SDF-1 and CXCR4, SHR exhibit a weaker, delayed response.

  4. Entrappment of alkaline protease and β-galactosidase in radiation stitched together poly-N-vinylcaprolactam

    International Nuclear Information System (INIS)

    Davidenko, T.I.; Kravchenko, I.A.

    1996-01-01

    The gel formations by poly-N-vinylcaprolactam upon its γ-irradiation by the 20-25 kGy dose as a results of partial polymer stitching together is shown, which is confirmed by the CD-and thermogravimetric data. By the alkaline protease and β-galactosidase entrapment in poly-N- vinylcaprolactam stitched together by γ-irradiation, the active preparations are obtained with 90-98 % and 30-35 % activity retained for alkaline protease and β-galactosidase, respectively. The increased stability of alkaline protease at acidic pH values and higher temperature was noted, and for β-galactosidase - the possibility of repeated use of the obtained preparation for lactose hydrolysis

  5. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    Science.gov (United States)

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  6. Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

    Directory of Open Access Journals (Sweden)

    Franziska Buch

    Full Text Available Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep. Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

  7. Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex.

    Science.gov (United States)

    Siritapetawee, J; Thammasirirak, S; Samosornsuk, W

    2012-01-01

    Artocarpus heterophyllus (jackfruit) is a latex producing plant. Plant latex is produced from secretory cells and contains many intergradients. It also has been used in folk medicine. This study aimed to purify and characterize the biological activities of a protease from jackfruit latex. A protease was isolated and purified from crude latex of a jackfruit tree by acid precipitation and ion exchange chromatography. The proteolytic activities of protein were tested using gelatin- and casein-zymography. The molecular weight and isoelectric point (pl) of protein were analysed by SDS/12.5% PAGE and 2D-PAGE, respectively. Antimicrobial activity of protein was analysed by broth microdilution method. In addition, the antibacterial activity of protein against Pseudomonas aeruginosa ATCC 27853 was observed and measured using atomic force microscopy (AFM) technique. The purified protein contained protease activity by digesting gelatin- and casein-substrates. The protease was designated as antimicrobial protease-48 kDa or AMP48 due to its molecular mass on SDS-PAGE was approximately 48 kDa. The isoelectric point (pl) of AMP48 was approximately 4.2. In addition, AMP48 contained antimicrobial activities by it could inhibit the growths of Pseudomonas aeruginosa ATCC 27853 and clinical isolated Candida albicans at minimum inhibitory concentration (MIC) 2.2 mg/ml and Minimum microbicidal concentration (MMC) 8.8 mg/ml. AFM image also supported the antimicrobial activities of AMP48 by the treated bacterial morphology and size were altered from normal.

  8. An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease.

    Science.gov (United States)

    Pan, Rong; Zhou, Yuan; He, Hai-Jin; He, Rong-Qiao

    2011-04-01

    The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5'-phosphate dsDNA (5'P DNA) and degrades it without sequence specificity, but does not recognize 5'OH DNA. As is the case for most DNases, Mg(2+) was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Production and partial characterization of proteases from Mucor hiemalis URM3773

    Directory of Open Access Journals (Sweden)

    Roana Cecília dos Santos Ribeiro

    2015-03-01

    Full Text Available The current study evaluated the proteases production from 11 fungal species belonging to the genera Mucor, Rhizomucor and Absidia. The species were obtained from the Collection of Cultures URM at the Mycology Department-UFPE, Brazil. The best producing species was Mucor hiemalis URM 3773 (1.689 U mL-1. Plackett-Burman design methodology was employed to select the most effective parameter for protease production out of 11 medium components, including: concentration of filtrate soybean, glucose, incubation period, yeast extract, tryptone, pH, aeration, rotation, NH4Cl, MgSO4 and K2HPO4. Filtrated soybean concentration was the significant variable over the response variable, which was the specific protease activity. The crude enzyme extract showed optimal activity in pH 7.5 and at 50ºC. The enzyme was stable within a wide pH range from 5.8 to 8.0, in the phosphate buffer 0.1M and in stable temperature variation of 40-70ºC, for 180 minutes. The ions FeSO4, NaCl, MnCl2, MgCl2 and KCl stimulated the protease activity, whereas ZnCl2 ion inhibited the activity in 2.27%. Iodoacetic acid at 1mM was the proteases inhibitor that presented greater action.The results indicate that the studied enzyme have great potential for industrial application.

  10. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I

    2001-01-01

    A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

  11. Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93.

    Science.gov (United States)

    Salihi, Ahsan; Asoodeh, Ahmad; Aliabadian, Mansour

    2017-01-01

    In this study, Aspergillus oryzae CH93 was isolated from soil sample and examined using molecular analysis. Following culture of A. oryzae CH93 under optimal enzyme production, a 47.5kDa extracellular protease was purified using ammonium sulfate precipitation and Q-Sepharose chromatography. The optimal pH 8 and temperature of 50°C obtained for the isolated protease. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), H 2 O 2 decreased activity, while Triton X-100 and phenylmethanesulfonyl fluoride (PMSF) had no inhibitory effect on the enzyme activity; meanwhile, 2-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA) declined the protease activity. Isoamyl alcohol and acetone (30%) enhanced activity whereas 2-propanol, isopropanol and dimethyl sulfoxide (DMSO) (30%) reduced protease activity. The enzyme exhibited a half-life of 100min at its optimum temperature. Among five substrates of bovine serum albumin (BSA), N-acetyl-l-tyrosine ethyl ester monohydrate (ATEE), casein, azocasein and gelatin results showed that casein is the best substrate with V max of 0.1411±0.004μg/min and K m of 2.432±0.266μg/ml. In conclusion, the extracted protease from A. oryzae CH93 as a fungal source possessed biochemical features which could be useful in some application usages. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Dietary fatty acids modulate associations between genetic variants and circulating fatty acids in plasma and erythrocyte membranes: meta-analysis of nine studies in the CHARGE consortium

    Science.gov (United States)

    Scope: Tissue concentrations of omega-3 fatty acids may reduce cardiovascular disease risk, and genetic variants are associated with circulating fatty acids concentrations. Whether dietary fatty acids interact with genetic variants to modify circulating omega-3 fatty acids is unclear. We evaluated i...

  13. Chemical and volatile composition of jujube wines fermented by Saccharomyces cerevisiae with and without pulp contact and protease treatment

    Directory of Open Access Journals (Sweden)

    Wenye ZHANG

    2016-01-01

    Full Text Available Abstract This study evaluated the chemical and volatile composition of jujube wines fermented with Saccharomyces cerevisiae A1.25 with and without pulp contact and protease treatment during fermentation. Yeast cell population, total reducing sugar and methanol contents had significant differences between nonextracted and extracted wine. The nonextracted wines had significantly higher concentrations of ethyl 9-hexadecenoate, ethyl palmitate and ethyl oleate than the extracted wines. Pulp contact also could enhance phenylethyl alcohol, furfuryl alcohol, ethyl palmitat and ethyl oleate. Furthermore, protease treatment can accelerate the release of fusel oils. The first principal component separated the wine from the extracted juice without protease from other samples based on the higher concentrations of medium-chain fatty acids and medium-chain ethyl esters. Sensory evaluation showed pulp contact and protease could improve the intensity and complexity of wine aroma due to the increase of the assimilable nitrogen.

  14. Multiple roles of the coagulation protease cascade during virus infection.

    Science.gov (United States)

    Antoniak, Silvio; Mackman, Nigel

    2014-04-24

    The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

  15. Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn.

    Science.gov (United States)

    Ferraretto, L F; Fredin, S M; Shaver, R D

    2015-10-01

    Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated

  16. Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

    Science.gov (United States)

    Choo, Young Moo; Lee, Kwang Sik; Yoon, Hyung Joo; Kim, Bo Yeon; Sohn, Mi Ri; Roh, Jong Yul; Je, Yeon Ho; Kim, Nam Jung; Kim, Iksoo; Woo, Soo Dong; Sohn, Hung Dae; Jin, Byung Rae

    2010-05-03

    Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

  17. Arachidonic Acid Metabolism Pathway Is Not Only Dominant in Metabolic Modulation but Associated With Phenotypic Variation After Acute Hypoxia Exposure

    Directory of Open Access Journals (Sweden)

    Chang Liu

    2018-03-01

    Full Text Available Background: The modulation of arachidonic acid (AA metabolism pathway is identified in metabolic alterations after hypoxia exposure, but its biological function is controversial. We aimed at integrating plasma metabolomic and transcriptomic approaches to systematically explore the roles of the AA metabolism pathway in response to acute hypoxia using an acute mountain sickness (AMS model.Methods: Blood samples were obtained from 53 enrolled subjects before and after exposure to high altitude. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and RNA sequencing were separately performed for metabolomic and transcriptomic profiling, respectively. Influential modules comprising essential metabolites and genes were identified by weighted gene co-expression network analysis (WGCNA after integrating metabolic information with phenotypic and transcriptomic datasets, respectively.Results: Enrolled subjects exhibited diverse response manners to hypoxia. Combined with obviously altered heart rate, oxygen saturation, hemoglobin, and Lake Louise Score (LLS, metabolomic profiling detected that 36 metabolites were highly related to clinical features in hypoxia responses, out of which 27 were upregulated and nine were downregulated, and could be mapped to AA metabolism pathway significantly. Integrated analysis of metabolomic and transcriptomic data revealed that these dominant molecules showed remarkable association with genes in gas transport incapacitation and disorders of hemoglobin metabolism pathways, such as ALAS2, HEMGN. After detailed description of AA metabolism pathway, we found that the molecules of 15-d-PGJ2, PGA2, PGE2, 12-O-3-OH-LTB4, LTD4, LTE4 were significantly up-regulated after hypoxia stimuli, and increased in those with poor response manner to hypoxia particularly. Further analysis in another cohort showed that genes in AA metabolism pathway such as PTGES, PTGS1, GGT1, TBAS1 et al. were excessively

  18. Modulating lignin in plants

    Science.gov (United States)

    Apuya, Nestor; Bobzin, Steven Craig; Okamuro, Jack; Zhang, Ke

    2013-01-29

    Materials and methods for modulating (e.g., increasing or decreasing) lignin content in plants are disclosed. For example, nucleic acids encoding lignin-modulating polypeptides are disclosed as well as methods for using such nucleic acids to generate transgenic plants having a modulated lignin content.

  19. Serine protease inhibitors of parasitic helminths.

    Science.gov (United States)

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

  20. Tunable protease-activatable virus nanonodes.

    Science.gov (United States)

    Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

    2014-05-27

    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

  1. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase

    Science.gov (United States)

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

    2014-01-01

    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca2+-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca2+ increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes. PMID:24799560

  2. Protease-activated receptor-2 activation exaggerates TRPV1-mediated cough in guinea pigs.

    Science.gov (United States)

    Gatti, Raffaele; Andre, Eunice; Amadesi, Silvia; Dinh, Thai Q; Fischer, Axel; Bunnett, Nigel W; Harrison, Selena; Geppetti, Pierangelo; Trevisani, Marcello

    2006-08-01

    A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.

  3. Modulation of hexavalent chromium toxicity on Οriganum vulgare in an acidic soil amended with peat, lime, and zeolite.

    Science.gov (United States)

    Antoniadis, Vasileios; Zanni, Anna A; Levizou, Efi; Shaheen, Sabry M; Dimirkou, Anthoula; Bolan, Nanthi; Rinklebe, Jörg

    2018-03-01

    Dynamics of chromate (Cr(VI)) in contaminated soils may be modulated by decreasing its phytoavailability via the addition of organic matter-rich amendments, which might accelerate Cr(VI) reduction to inert chromite (Cr(III)) or high-cation exchange capacity amendments. We studied Cr(VI) phytoavailability of oregano in a Cr(VI)-spiked acidic soil non-treated (S) and treated with peat (SP), lime (SL), and zeolite (SZ). The addition of Cr(VI) increased the concentrations of Cr(VI) and Cr(III) in soils and plants, especially in the lime-amended soil. The plant biomass decreased in the lime-amended soil compared to the un-spiked soil (control) due to decreased plant phosphorus concentrations and high Cr(VI) concentrations in root at that treatment. Oregano in the peat-amended soil exhibited significantly less toxic effects, due to the role of organic matter in reducing toxic Cr(VI) to Cr(III) and boosted plant vigour in this treatment. In the lime-amended soil, the parameters of soil Cr(VI), soil Cr(III), and root Cr(III) increased significantly compared to the non-amended soil, indicating that Cr(VI) reduction to Cr(III) was accelerated at high pH. Added zeolite failed to decreased Cr(VI) level to soil and plant. Oregano achieved a total uptake of Cr(III) and Cr(VI) of 0.275 mg in plant kg -1 soil in a pot in the non-amended soil. We conclude that peat as soil amendment might be considered as a suitable option for decreasing Cr(VI) toxicity in soil and plant, and that oregano as tolerant plant species has a certain potential to be used as a Cr accumulator. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jin Kyeong [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Molecular Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Kim, Sung-Wan; Kim, Duk-Sil [Department of Thoracic and Cardiovascular Surgery, CHA Gumi Medical Center, CHA University, Gumi 730-040 (Korea, Republic of); Lee, Jong Yeong [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Lee, Soyoung [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Bio-Materials Research Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 580-185 (Korea, Republic of); Oh, Hyun-Mee [Bio-Materials Research Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 580-185 (Korea, Republic of); Ha, Yeong Su; Yoo, Jeongsoo [Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Park, Pil-Hoon [College of Pharmacy, Yeungnam University, Gyeongbuk 712-749 (Korea, Republic of); Shin, Tae-Yong [College of Pharmacy, Woosuk University, Jeonju 565-701 (Korea, Republic of); Kwon, Taeg Kyu [Department of Immunology, School of Medicine, Keimyung University, Daegu 704-701 (Korea, Republic of); Rho, Mun-Chual, E-mail: rho-m@kribb.re.kr [Bio-Materials Research Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 580-185 (Korea, Republic of); Kim, Sang-Hyun, E-mail: shkim72@knu.ac.kr [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2016-01-01

    ABSTRACT: Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with a combination of synovium joint inflammation, synovium hyperplasia, and destruction of cartilage and bone. Oleanolic acid acetate (OAA), a compound isolated from Vigna angularis, has been known to possess pharmacological activities, including anti-inflammation and anti-bone destruction. In this study, we investigated the effects of OAA on RA and the underlying mechanisms of action by using a type-II collagen-induced arthritis (CIA) mouse model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. Oral administration of OAA decreased the clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum total and anti-type II collagen IgG, IgG1, and IgG2a levels. OAA administration reduced Th1/Th17 phenotype CD4{sup +} T lymphocyte expansions and inflammatory cytokine productions in T cell activated draining lymph nodes and spleen. OAA reduced the expression and production of inflammatory mediators, such as cytokines and matrix metalloproteinase (MMP)-1/3, in the ankle joint tissue and RA synovial fibroblasts by down-regulating Akt, mitogen-activated protein kinases, and nuclear factor-κB. Our results clearly support that OAA plays a therapeutic role in RA pathogenesis by modulating helper T cell immune responses and matrix-degrading enzymes. The immunosuppressive effects of OAA were comparable to dexamethasone and ketoprofen. We provide evidences that OAA could be a potential therapeutic candidate for RA. - Highlights: • OAA attenuated chronic CIA symptoms. • OAA had a regulating effect on the T helper cell immune reaction for CIA. • The effect of OAA on the RA was comparable to the dexamethasone or ketoprofen. • OAA might be a candidate for the treatment of arthritic diseases.

  5. Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog*

    Science.gov (United States)

    Mulligan, Christopher; Mindell, Joseph A.

    2013-01-01

    Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na+-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, GltPh, suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3–4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, GltPh is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved GltPh retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process. PMID:24155238

  6. Modulating the nanorods protrusion from poly(allylamine hydrochloride)-g-pyrene microcapsules by 1-pyrenesulfonic acid sodium salt.

    Science.gov (United States)

    Guan, Erjia; Wang, Tianxiang; Wang, Zhipeng; Gao, Changyou

    2013-09-01

    It was found previously that the Schiff base bonds in poly(allylamine hydrochloride)-g-pyrene (PAH-Py) microcapsules (MCs) are hydrolyzed at pH 2 within 1 h, leading to disassembly of the MCs and protrusion of pyrene aldehyde (Py) nanorods (NRs) on the capsule surface. Herein, we found a new way to modulate the protrusion of NRs by addition of 1-pyrenesulfonic acid sodium salt (PySO3Na). Along with the increase in PySO3Na to Py molar ratio in the MCs solution, the protrusion of NRs was progressively blocked and even inhibited at a ratio of 2.3, and at this condition, the microcapsules were stable under pH 2 for 24 h. After the composite microcapsules with excess PySO3Na were washed with a pH 10 solution and then incubated in a pH 2 solution, the NRs could be protruded from the MCs again. The fluorescence peak position of the PAH-Py/PySO3Na MCs gradually red-shifted with a decrease in pH value, and a sharp transition occurred at p H3.6, demonstrating the formation of pyrene excimers between the PySO3Na small molecules and the pendant Py groups on the PAH chain. The formed excimers take the role of blocking the self-assembly of cleaved Py molecules instead of inhibiting the hydrolysis of the Schiff base, whereas the MCs were stabilized by the charge interaction between PySO3Na and PAH backbone and the hydrophobic interaction between the pyrene rings. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids

    DEFF Research Database (Denmark)

    Bergenholm, David; Gossing, Michael; Wei, Yongjun

    2018-01-01

    Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food additives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics...... as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids...... industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature...

  8. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity. ... Generally, alkaline proteases are produced using submerged fermentation .... biopolymer concentrations were reported to have an influence ... adding nitrogenous compounds stimulate microorganism growth and ...

  9. Optimization of alkaline protease production and its fibrinolytic ...

    African Journals Online (AJOL)

    Optimization of alkaline protease production and its fibrinolytic activity from the ... nitrogen sources and sodium chloride concentration for protease production by the ... exploited to assist in protein degradation in various industrial processes.

  10. Purification and characterization of protease from Bacillus cereus ...

    African Journals Online (AJOL)

    Among them, SU12 isolate was selected due to its high enzyme production ... growth and protease production which includes different carbon and nitrogen sources, ... organism for the industrial production of the extracellular protease enzyme.

  11. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  12. Covalent glycoinositolphospholipid (GPI binding to hemoglobin is associated with insulin-activation of erythrocyte membrane protease

    Directory of Open Access Journals (Sweden)

    VESNA NIKETIC

    2004-05-01

    Full Text Available Recently, it was demonstrated that prolonged hyperinsulinism associated with hypoglycemia, both in vivo and in vitro, caused covalent glycoinositolphospholipid (GPI binding to the C termini of both hemoglobin b-chains, which resulted in the formation of a novel, hitherto unrecognized, minor hemoglobin fraction (GPI-Hb (Niketic et al., Biochem. Biophys. Res. Commun. 239 (1997 435. In this study it was demonstrated that exposure of erythrocyte membranes to insulin causes the activation of membrane protease as well as that the formation of GPI-Hb parallels its activity. It is suggested that the insulin-activated protease is able to catalyze, albeit slowly, the transpeptidation, i.e., the replacement of the carboxy-terminal amino acid(s residues of the Hb b-chains with GPI as an exogenous nucleophile. To our knowledge the present results show for the first time that insulin stimulates protease activity in erythrocyte membranes, as well as that insulin-activated protease may be involved in post-translational GPI binding to proteins.

  13. Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

    Directory of Open Access Journals (Sweden)

    Shoba Subramanian

    Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

  14. Modulation of Intestinal Barrier and Bacterial Endotoxin Production Contributes to the Beneficial Effect of Nicotinic Acid on Alcohol-Induced Endotoxemia and Hepatic Inflammation in Rats

    Directory of Open Access Journals (Sweden)

    Wei Zhong

    2015-10-01

    Full Text Available Alcohol consumption causes nicotinic acid deficiency. The present study was undertaken to determine whether dietary nicotinic acid supplementation provides beneficial effects on alcohol-induced endotoxin signaling and the possible mechanisms at the gut-liver axis. Male Sprague-Dawley rats were pair-fed the Lieber-DeCarli liquid diets containing ethanol or isocaloric maltose dextrin for eight weeks, with or without dietary supplementation with 750 mg/liter nicotinic acid. Chronic alcohol feeding elevated the plasma endotoxin level and activated hepatic endotoxin signaling cascade, which were attenuated by nicotinic acid supplementation. Alcohol consumption remarkably decreased the mRNA levels of claudin-1, claudin-5, and ZO-1 in the distal intestine, whereas nicotinic acid significantly up-regulated these genes. The concentrations of endotoxin, ethanol, and acetaldehyde in the intestinal contents were increased by alcohol exposure, and niacin supplementation reduced the intestinal endotoxin and acetaldehyde levels. Nicotinic acid supplementation upregulated the intestinal genes involved in aldehyde detoxification via transcriptional regulation. These results demonstrate that modulation of the intestinal barrier function and bacterial endotoxin production accounts for the inhibitory effects of nicotinic acid on alcohol-induced endotoxemia and hepatic inflammation.

  15. Additional file 5: Figure S1. of Uncovering co-expression gene network modules regulating fruit acidity in diverse apples

    OpenAIRE

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Zhong, Gan-Yuan; Xu, Kenong

    2015-01-01

    Analysis of modules Black, Brown, Blue and Yellow. (A) Module eigengene values across the 29 samples, including 17 in Ma_ on left and 12 in mama on right. Samples are represented by the combination of a letter (abbreviated cultivar name) and a number (replicate) (see legends in Fig. 1, 4 for keys). (B) Correlation between module membership (MM) and gene significance (GS) for malate. (PPTX 75 kb)

  16. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

    Directory of Open Access Journals (Sweden)

    Rajesh Prasad

    Full Text Available Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4. Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5, suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3 to moderate (KP4 preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease

  17. Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

    Science.gov (United States)

    Marchand, Sophie; Vandriesche, Gonzalez; Coorevits, An; Coudijzer, Katleen; De Jonghe, Valerie; Dewettinck, Koen; De Vos, Paul; Devreese, Bart; Heyndrickx, Marc; De Block, Jan

    2009-07-31

    Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

  18. Specificity and Application of the Lantibiotic Protease NisP

    Directory of Open Access Journals (Sweden)

    Manuel Montalbán-López

    2018-02-01

    Full Text Available Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

  19. Pengaruh PH dan Suhu terhadap Aktivitas Protease Penicillium SP.

    OpenAIRE

    Yusriah, Yusriah; Kuswytasari, Nengah Dwianita

    2013-01-01

    Tujuan penelitian ini adalah untuk mengetahui pengaruh pH dan suhu terhadap aktivitas protease pada Penicillium sp.3 T3f2. Selanjutnya, isolat Penicillium sp. di kultur dalam media produksi protease untuk menghasilkan protease. Suhu yang digunakan adalah 300 – 500C sedangkan pH-nya 4 – 8. Aktivitas protease ditentukan dan diukur dengan spektrofotometer pada panjang gelombang 275 nm, dengan kasein sebagai substrat. Berdasarkan uji ANOVA yang dilanjutkan dengan uji Duncan dengan taraf kepercaya...

  20. Phenolic acid intake, delivered via moderate champagne wine consumption, improves spatial working memory via the modulation of hippocampal and cortical protein expression/activation.

    Science.gov (United States)

    Corona, Giulia; Vauzour, David; Hercelin, Justine; Williams, Claire M; Spencer, Jeremy P E

    2013-11-10

    While much data exist for the effects of flavonoid-rich foods on spatial memory in rodents, there are no such data for foods/beverages predominantly containing hydroxycinnamates and phenolic acids. To address this, we investigated the effects of moderate Champagne wine intake, which is rich in these components, on spatial memory and related mechanisms relative to the alcohol- and energy-matched controls. In contrast to the isocaloric and alcohol-matched controls, supplementation with Champagne wine (1.78 ml/kg BW, alcohol 12.5% vol.) for 6 weeks led to an improvement in spatial working memory in aged rodents. Targeted protein arrays indicated that these behavioral effects were paralleled by the differential expression of a number of hippocampal and cortical proteins (relative to the isocaloric control group), including those involved in signal transduction, neuroplasticity, apoptosis, and cell cycle regulation. Western immunoblotting confirmed the differential modulation of brain-derived neurotrophic factor, cAMP response-element-binding protein (CREB), p38, dystrophin, 2',3'-cyclic-nucleotide 3'-phosphodiesterase, mammalian target of rapamycin (mTOR), and Bcl-xL in response to Champagne supplementation compared to the control drink, and the modulation of mTOR, Bcl-xL, and CREB in response to alcohol supplementation. Our data suggest that smaller phenolics such as gallic acid, protocatechuic acid, tyrosol, caftaric acid, and caffeic acid, in addition to flavonoids, are capable of exerting improvements in spatial memory via the modulation in hippocampal signaling and protein expression. Changes in spatial working memory induced by the Champagne supplementation are linked to the effects of absorbed phenolics on cytoskeletal proteins, neurotrophin expression, and the effects of alcohol on the regulation of apoptotic events in the hippocampus and cortex.

  1. Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

    Science.gov (United States)

    Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y

    2000-10-31

    Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

  2. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    Science.gov (United States)

    2009-01-01

    Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co

  3. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2009-03-01

    Full Text Available Abstract Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW and LexA (hoxW. In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer

  4. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

    Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

    2003-01-01

    of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...

  5. HIV-1 protease-induced apoptosis

    Czech Academy of Sciences Publication Activity Database

    Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, T.

    2014-01-01

    Roč. 11, May 20 (2014), 37/1-37/15 ISSN 1742-4690 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : HIV protease * BCA3 * AKIP-1 * apoptosis * mitochondria Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/37

  6. Bacterial proteases: targets for diagnostics and therapy

    NARCIS (Netherlands)

    Kaman, W.E.; Hays, J.P.; Endtz, H.P.; Bikker, F.J.

    2014-01-01

    Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and

  7. Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids.

    Science.gov (United States)

    Bergenholm, David; Gossing, Michael; Wei, Yongjun; Siewers, Verena; Nielsen, Jens

    2018-04-01

    Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food additives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized. We engineered Saccharomyces cerevisiae yeast strains toward a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type. © 2018 Wiley Periodicals, Inc.

  8. Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

    Directory of Open Access Journals (Sweden)

    M Holzhausen

    2005-03-01

    Full Text Available Proteinase-activated receptor-2 (PAR2 belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

  9. Enhancing emulsification and antioxidant ability of egg albumin by moderately acid hydrolysis: Modulating an emulsion-based system for mulberry seed oil.

    Science.gov (United States)

    Chang, Jing; Kang, Xu; Yuan, Jiang-Lan

    2018-07-01

    Mulberry seed oil (MSO) is a kind of potential health-care lipids. This study, we investigated unsaturated fatty acids profiles of freshly squeezed MSO by GC-MS and modulated an oil-in-water emulsion system stabilized by acid hydrolyzed egg albumin (AHEA) to protect MSO from oxidation. The results showed that the content of total unsaturated fatty acids in MSO was almost 80%, of which 9, 12- and 10, 13-linoleic acid was over 60% and 10% respectively. In the case of the MSO-in-AHEA emulsions, it was observed that acid hydrolysis improved emulsifying effect, emulsifying stability and antioxidant activity of egg albumin (EA). The hydrolysates of EA (1%, w/w) acid hydrolyzed for 4 h at 85 °C had the best DPPH radical scavenging efficiency. It was suitable for EA to hydrolyze for 4 to 12 h at pH 2.5 and 85 °C because of their better emulsification and oxidation stability than the others. The results about AHEA could be valuable for designing delivery and protect systems for MSO or other bioactive component to avoid their oxidative damage or control their release. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. A novel protease activity assay using a protease-responsive chaperone protein

    International Nuclear Information System (INIS)

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-01-01

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  11. A novel protease activity assay using a protease-responsive chaperone protein

    Energy Technology Data Exchange (ETDEWEB)

    Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  12. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

    Science.gov (United States)

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  13. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Luca Musante

    2015-01-01

    Full Text Available Diabetic nephropathy (DN is one of the major complications of diabetes mellitus (DM, leads to chronic kidney disease (CKD, and, ultimately, is the main cause for end-stage kidney disease (ESKD. Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  14. Rapid method for measuring protease activity in milk using radiolabeled casein

    International Nuclear Information System (INIS)

    Christen, G.L.

    1987-01-01

    A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl- 14 C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations of substrate from 2 to 20 nCi gave counts per minute, which increased linearly when counted with the Charm analyzer. There was not a significant difference in counting times of 10, 20, or 30 min. A mixture of sodium acetate and acetic acid precipitated nonhydrolyzed substrate with an efficiency of 97%. Comparison of the [ 14 C] casein assay, a casein fluorescein isothiocyanate assay, trinitrobenzenesulfonic acid procedure, and the Hull procedure using protease from psychrotrophic bacteria revealed that the [ 14 C] casein and casein fluorescein isothiocyanate methods were roughly equivalent and that the radiometric procedure was 10 times more sensitive than the trinitrobenzenesulfonic acid assay. The radiometric procedure was approximately 10(4) times more sensitive than the Hull procedure. The [ 14 C] casein and casein fluorescein isothiocyanate methods were similar in time required, about 30 min, while the trinitrobenzenesulfonic acid assay and Hull method required about 1 h plus reagent preparation time. The [ 14 C] casein procedure was most expensive per test; the other three were cheaper and similar to each other in cost

  15. Photothermal-modulated drug delivery and magnetic relaxation based on collagen/poly(γ-glutamic acid hydrogel

    Directory of Open Access Journals (Sweden)

    Cho SH

    2017-03-01

    Full Text Available Sun-Hee Cho,1,* Ahreum Kim,1,* Woojung Shin,2 Min Beom Heo,1 Hyun Jong Noh,1 Kwan Soo Hong,3,4 Jee-Hyun Cho,3,4 Yong Taik Lim1,2 1SKKU Advanced Institute of Nanotechnology (SAINT, 2School of Chemical Engineering, Sungkyunkwan University, Suwon, 3Bioimaging Research Team, Korea Basic Science Institute, Cheongju, 4Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea *These authors contributed equally to this work Abstract: Injectable and stimuli-responsive hydrogels have attracted attention in molecular imaging and drug delivery because encapsulated diagnostic or therapeutic components in the hydrogel can be used to image or change the microenvironment of the injection site by controlling various stimuli such as enzymes, temperature, pH, and photonic energy. In this study, we developed a novel injectable and photoresponsive composite hydrogel composed of anticancer drugs, imaging contrast agents, bio-derived collagen, and multifaceted anionic polypeptide, poly (γ-glutamic acid (γ-PGA. By the introduction of γ-PGA, the intrinsic temperature-dependent phase transition behavior of collagen was modified to a low viscous sol state at room temperature and nonflowing gel state around body temperature. The modified temperature-dependent phase transition behavior of collagen/γ-PGA hydrogels was also evaluated after loading of near-infrared (NIR fluorophore, indocyanine green (ICG, which could transform absorbed NIR photonic energy into thermal energy. By taking advantage of the abundant carboxylate groups in γ-PGA, cationic-charged doxorubicin (Dox and hydrophobic MnFe2O4 magnetic nanoparticles were also incorporated successfully into the collagen/γ-PGA hydrogels. By illumination of NIR light on the collagen/γ-PGA/Dox/ICG/MnFe2O4 hydrogels, the release kinetics of Dox and magnetic relaxation of MnFe2O4 nanoparticles could be modulated. The experimental results suggest that

  16. TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

    Science.gov (United States)

    Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

    2014-08-03

    Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

  17. HIV protease drug resistance and its impact on inhibitor design.

    Science.gov (United States)

    Ala, P J; Rodgers, J D; Chang, C H

    1999-07-01

    The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

  18. Development of potent inhibitors of the coxsackievirus 3C protease

    International Nuclear Information System (INIS)

    Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin; Kim, Yong-Chul

    2007-01-01

    Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro

  19. HIDROLISIS IKAN BERNILAI EKONOMI RENDAH SECARA ENZIMATIS MENGGUNAKAN PROTEASE BIDURI [Enzymatic Hydrolysis of Low Economic Value Fishes using Biduri’s Protease

    Directory of Open Access Journals (Sweden)

    Yuli Witono1*

    2015-07-01

    Full Text Available Fish protein hydrolyzate is a product obtained from the decomposition of fish proteins into short-chain compounds due to the hydrolysis process either by enzymes, acids, or bases. The purpose of this study was to optimize the production of fish protein hydrolyzate from the low economic value fishes including 'bibisan' (Apogon albimaculosus, 'baji-baji' (Platycephalidae cymbacephalus, and the 'lidah' (Cynoglossus lingua obtained from Talango Island, Madura. Production of fish protein hydrolyzate was conducted using (Calotropis gigantea protease at various concentrations (0, 0.7, 1.4, and 2.1 Unit/g and at various hydrolysis times (0, 1.5, and 3 h. The experimental design was a Completely Randomized Design with two factors and the experiments were carried out in triplicates. The results showed that interactions between protease concentrations and hydrolysis times significantly affected (at 5% level test the soluble proteins, maillard products, and the level of rancidity measured as thiobarbituric acid (TBA. The best fish hydrolyzate product based on the soluble protein parameter was resulted from 2.1 unit/g of biduri’s protease with a hydrolysis time of 1.5 h. The hydrolyzate produced had 3.51% of soluble proteins, a maillard value of 0.63, and rancidity levels of 12.21 mmol TBA/kg.

  20. Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

    Science.gov (United States)

    Ibrahim, Ahmed Ragaa Nour; Kawamoto, Seiji; Aki, Tsunehiro; Shimada, Yayoi; Rikimaru, Satoshi; Onishi, Nobukazu; Babiker, Elfadil Elfadl; Oiso, Isao; Hashimoto, Kunihiko; Hayashi, Takaharu; Ono, Kazuhisa

    2010-01-01

    Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis. 2010 S. Karger AG, Basel.

  1. Pathophysiological significance and therapeutic applications of snake venom protease inhibitors.

    Science.gov (United States)

    Thakur, Rupamoni; Mukherjee, Ashis K

    2017-06-01

    Protease inhibitors are important constituents of snake venom and play important roles in the pathophysiology of snakebite. Recently, research on snake venom protease inhibitors has provided valuable information to decipher the molecular details of various biological processes and offer insight for the development of some therapeutically important molecules from snake venom. The process of blood coagulation and fibrinolysis, in addition to affecting platelet function, are well known as the major targets of several snake venom protease inhibitors. This review summarizes the structure-functional aspects of snake venom protease inhibitors that have been described to date. Because diverse biological functions have been demonstrated by protease inhibitors, a comparative overview of their pharmacological and pathophysiological properties is also highlighted. In addition, since most snake venom protease inhibitors are non-toxic on their own, this review evaluates the different roles of individual protease inhibitors that could lead to the identification of drug candidates and diagnostic molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Tissue Fatty Acid Profile is Differently Modulated from Olive Oil and Omega-3 Polyunsaturated Fatty Acids in ApcMin/+ Mice.

    Science.gov (United States)

    Tutino, Valeria; Caruso, Maria G; De Leonardis, Giampiero; De Nunzio, Valentina; Notarnicola, Maria

    2017-11-16

    Fatty acid profile can be considered an appropriate biomarker for investigating the relations between the patterns of fatty acid metabolism and specific diseases, as cancer, cardiovascular and degenerative diseases. Aim of this study was to test the effects of diets enriched with olive oil and omega-3 Polyunsaturated Fatty Acids (PUFAs) on fatty acid profile in intestinal tissue of ApcMin/+ mice. Three groups of animals were considered: control group, receiving a standard diet; olive oilgroup, receiving a standard diet enriched with olive oil; omega-3 group, receiving a standard diet enriched with salmon fish. Tissue fatty acid profile was evaluated by gas chromatography method. Olive oil and omega-3 PUFAs in the diet differently affect the tissue fatty acid profile. Compared to control group, the levels of Saturated Fatty Acids (SFAs) were lower in olive oil group, while an increase of SFAs was found in omega-3 group. Monounsaturated Fatty Acids (MUFAs) levels were enhanced after olive oil treatment, and in particular, a significant increase of oleic acid levels was detected; MUFAs levels were instead reduced in omega-3 group in line with the decrease of oleic acid levels. The total PUFAs levels were lower in olive oil respect to control group. Moreover, a significant induction of Saturation Index (SI) levels was observed after omega-3 PUFAs treatment, while its levels were reduced in mice fed with olive oil. Our data demonstrated a different effect of olive oil and omega-3 PUFAs on tissue lipid profile in APCMin/+ mice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

    Science.gov (United States)

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

    2010-12-01

    The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.

  4. Tumour–stromal interactions in acid-mediated invasion: A mathematical model

    KAUST Repository

    Martin, Natasha K.

    2010-12-01

    It is well established that the tumour microenvironment can both promote and suppress tumour growth and invasion, however, most mathematical models of invasion view the normal tissue as inhibiting tumour progression via immune modulation or spatial constraint. In particular, the production of acid by tumour cells and the subsequent creation of a low extracellular pH environment has been explored in several \\'acid-mediated tumour invasion\\' models where the acidic environment facilitates normal cell death and permits tumour invasion. In this paper, we extend the acid-invasion model developed by Gatenby and Gawlinski (1996) to include both the competitive and cooperative interactions between tumour and normal cells, by incorporating the influence of extracellular matrix and protease production at the tumour-stroma interface. Our model predicts an optimal level of tumour acidity which produces both cell death and matrix degradation. Additionally, very aggressive tumours prevent protease production and matrix degradation by excessive normal cell destruction, leading to an acellular (but matrix filled) gap between the tumour and normal tissue, a feature seen in encapsulated tumours. These results suggest, counterintuitively, that increasing tumour acidity may, in some cases, prevent tumour invasion.

  5. Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids.

    Science.gov (United States)

    Sen, Suranjana; Sirobhushanam, Sirisha; Johnson, Seth R; Song, Yang; Tefft, Ryan; Gatto, Craig; Wilkinson, Brian J

    2016-01-01

    The fatty acid composition of membrane glycerolipids is a major determinant of Staphylococcus aureus membrane biophysical properties that impacts key factors in cell physiology including susceptibility to membrane active antimicrobials, pathogenesis, and response to environmental stress. The fatty acids of S. aureus are considered to be a mixture of branched-chain fatty acids (BCFAs), which increase membrane fluidity, and straight-chain fatty acids (SCFAs) that decrease it. The balance of BCFAs and SCFAs in USA300 strain JE2 and strain SH1000 was affected considerably by differences in the conventional laboratory medium in which the strains were grown with media such as Mueller-Hinton broth and Luria broth resulting in high BCFAs and low SCFAs, whereas growth in Tryptic Soy Broth and Brain-Heart Infusion broth led to reduction in BCFAs and an increase in SCFAs. Straight-chain unsaturated fatty acids (SCUFAs) were not detected. However, when S. aureus was grown ex vivo in serum, the fatty acid composition was radically different with SCUFAs, which increase membrane fluidity, making up a substantial proportion of the total (37%) and BCFAs (>36%) making up the rest. Staphyloxanthin, an additional major membrane lipid component unique to S. aureus, tended to be greater in content in cells with high BCFAs or SCUFAs. Cells with high staphyloxanthin content had a lower membrane fluidity that was attributed to increased production of staphyloxanthin. S. aureus saves energy and carbon by utilizing host fatty acids for part of its total fatty acids when growing in serum, which may impact biophysical properties and pathogenesis given the role of SCUFAs in virulence. The nutritional environment in which S. aureus is grown in vitro or in vivo in an infection is likely to be a major determinant of membrane fatty acid composition.

  6. Activity, specificity, and probe design for the smallpox virus protease K7L.

    Science.gov (United States)

    Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S

    2012-11-16

    The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

  7. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    Science.gov (United States)

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques

    Directory of Open Access Journals (Sweden)

    Workman Chris

    2011-04-01

    Full Text Available Abstract Background Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer's yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile. Results In the present study, transcriptome and metabolome analysis were used to elucidate the effect on the addition of the multicomponent protease enzyme Flavourzyme and its influence on the metabolism of the brewer's yeast strain Weihenstephan 34/70. The study underlines the importance of sufficient nitrogen availability during the course of beer fermentation. The applied metabolome and transcriptome analysis allowed mapping the effect of the wort sugar composition on the nitrogen uptake. Conclusion Both the transcriptome and the metabolome analysis revealed that there is a significantly higher impact of protease addition for maltose syrup supplemented fermentations, while addition of glucose syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition.

  9. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

  10. Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

    Science.gov (United States)

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2015-10-15

    Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

  11. The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

    Science.gov (United States)

    Rosen, G; Shoshani, M; Naor, R; Sela, M N

    2001-12-01

    A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

  12. Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Park, J.H.; Lee, Y.S.; Chung, C.H.; Goldberg, A.L.

    1988-02-01

    Protease Re, a new cytoplasmic endoprotease in Escherichia coli, was purified to homogeneity by conventional procedures, using (/sup 3/H) casein as the substrate. The enzyme consists of a single polypeptide of 82,000 molecular weight. It is maximally active between pH 7 and 8.5 and is independent of ATP. It has a pI of 6.8 and a K/sub m/ of 10.8 ..mu..M for casein. Since diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride inhibited this enzyme, it appears to be a serine protease. Protease Re was sensitive to inhibition by L-1-tosylamido-2-phenylethylchloromethylketone but not to that by 1-chloro-3-tosylamido-7-aminoheptanone, thiol-blocking reagents, chelating agents, or various peptide aldehydes. Re also degraded (/sup 125/I) globin, (/sup 125/I) glucagon, and /sup 125/I-labeled denatured bovine serum albumin to acid-soluble products (generally oligopeptides of > 1500 daltons), but it showed no activity against serum albumin, growth hormone, insulin, or a variety of fluorometric peptide substrates. It also hydrolyzed oxidatively inactivated glutamine synthetase (generated by ascorbate, oxygen, and iron) four- to fivefold more rapidly than the native protein. Protease Re appears to be identical to the proteolytic enzyme isolated by Roseman and Levine by its ability to degrade selectively oxidatively damaged glutamine synthetase in vivo. Its role in intracellular protein breakdown is uncertain.

  13. Foliar application of amino acids modulates aroma components of 'FUJI' apple (malus domestica L.)

    International Nuclear Information System (INIS)

    Gou, W.; Zhang, L.; Chen, F.; Cui, Z.; Zhao, Y.; Zheng, P.; Tian, L.; Zhang, L.; Zhang, C.

    2015-01-01

    Volatile flavor compounds play a key role in determining the perception and acceptability as well as enhancing market competitiveness of apple (Malus domestica L.). In our study, we evaluated the effects of foliar-applied four different amino acids, i.e. leucine (Leu), isoleucine (Ile), valine (Val) and alanine (Ala), on aroma components and two key enzymes activities involved in aroma metabolism of Fuji apple. The total amount of aromatic components under Ala treatment was significantly higher than those under other treatments. There was a considerable increase in total aroma content, including hexanal, 2-methyl-butanol, nonanal, (E)-2-hexenal, methyleugenol, ethyl acetate, butanoic acid-pentyl ester, butanoic acid-hexyl ester, butyric acid ethyl ester, acetic acid-2-methyl-butyl ester, treated with spraying amino acids compared with the control. More specifically, hexanal, 2-methyl-butanol, methyleugenol and acetic acid-2-methyl-butyl ester exhibited a greater substantial increase of their contents than those of in other ingredients. However, butanoic acid-2-methyl-2-methyl butyl ester maintained a highest level among all aroma components regardless of different amino acids application. Furthermore, the activities of alcohol dehydrogenase (ADH) and alcohol acyltransferase (AAT) were much higher under Ala treatment than those under other treatments. We concluded that foliar-applied organic nitrogen (N), especially for Ala, can improve aroma metabolism and it could be used in production to enhance fruit quality on a commercial scale. (author)

  14. Modulation of salivation and heartburn in response to the site of acid infusion in the human oesophagus.

    Science.gov (United States)

    Dutta, S K; Agrawal, K; Mahmoud, M A

    2010-09-01

    The pathogenesis of gastro-oesophageal reflux disease includes increased acid reflux, reduced salivation and impaired peristalsis. This may depend upon the height of acid wave and magnitude of oesophageal mucosal exposure. Interestingly, the effect of site of acid infusion upon salivary secretion and heartburn has not been examined in any detail. To examine whether acid infusion in the upper oesophagus may cause increased salivation and heartburn as compared with acid infusion in the lower oesophagus. Twelve healthy male subjects (mean age 30) received infusions of HCl, citric acid and acetic acid at 10 and 20 cm above the lower oesophageal sphincter (LES) for fixed time periods. Parotid saliva collected periodically and heartburn severity scored using standardized scale. Standard statistical methods (paired t-tests, analysis of variance) were used to determine the significance of results. Acid infusion in the upper oesophagus increased parotid flow rate as compared with that in the lower oesophagus (P heartburn score at 20 cm as well as 10 cm above LES (P heartburn in response to acid infusion in the upper vs. lower part of the oesophagus. 2010 Blackwell Publishing Ltd.

  15. Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

    Science.gov (United States)

    Lin, Hailan; Lin, Xijian; Zhu, Jiwei; Yu, Xiao-Qiang; Xia, Xiaofeng; Yao, Fengluan; Yang, Guang; You, Minsheng

    2017-02-14

    Serine protease inhibitors (SPIs) have been found in all living organisms and play significant roles in digestion, development and innate immunity. In this study, we present a genome-wide identification and expression profiling of SPI genes in the diamondback moth, Plutella xylostella (L.), a major pest of cruciferous crops with global distribution and broad resistance to different types of insecticides. A total of 61 potential SPI genes were identified in the P. xylostella genome, and these SPIs were classified into serpins, canonical inhibitors, and alpha-2-macroglobulins based on their modes of action. Sequence alignments showed that amino acid residues in the hinge region of known inhibitory serpins from other insect species were conserved in most P. xylostella serpins, suggesting that these P. xylostella serpins may be functionally active. Phylogenetic analysis confirmed that P. xylostella inhibitory serpins were clustered with known inhibitory serpins from six other insect species. More interestingly, nine serpins were highly similar to the orthologues in Manduca sexta which have been demonstrated to participate in regulating the prophenoloxidase activation cascade, an important innate immune response in insects. Of the 61 P.xylostella SPI genes, 33 were canonical SPIs containing seven types of inhibitor domains, including Kunitz, Kazal, TIL, amfpi, Antistasin, WAP and Pacifastin. Moreover, some SPIs contained additional non-inhibitor domains, including spondin_N, reeler, and other modules, which may be involved in protein-protein interactions. Gene expression profiling showed gene-differential, stage- and sex-specific expression patterns of SPIs, suggesting that SPIs may be involved in multiple physiological processes in P. xylostella. This is the most comprehensive investigation so far on SPI genes in P. xylostella. The characterized features and expression patterns of P. xylostella SPIs indicate that the SPI family genes may be involved in innate immunity

  16. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  17. Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

    Science.gov (United States)

    Welsch, Ralf; Zhou, Xiangjun; Yuan, Hui; Álvarez, Daniel; Sun, Tianhu; Schlossarek, Dennis; Yang, Yong; Shen, Guoxin; Zhang, Hong; Rodriguez-Concepcion, Manuel; Thannhauser, Theodore W; Li, Li

    2018-01-08

    Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  18. Body Position Modulates Gastric Emptying and Affects the Post-Prandial Rise in Plasma Amino Acid Concentrations Following Protein Ingestion in Humans

    Directory of Open Access Journals (Sweden)

    Andrew M. Holwerda

    2016-04-01

    Full Text Available Dietary protein digestion and amino acid absorption kinetics determine the post-prandial muscle protein synthetic response. Body position may affect gastrointestinal function and modulate the post-prandial rise in plasma amino acid availability. We aimed to assess the impact of body position on gastric emptying rate and the post-prandial rise in plasma amino acid concentrations following ingestion of a single, meal-like amount of protein. In a randomized, cross-over design, eight healthy males (25 ± 2 years, 23.9 ± 0.8 kg·m−2 ingested 22 g protein and 1.5 g paracetamol (acetaminophen in an upright seated position (control and in a −20° head-down tilted position (inversion. Blood samples were collected during a 240-min post-prandial period and analyzed for paracetamol and plasma amino acid concentrations to assess gastric emptying rate and post-prandial amino acid availability, respectively. Peak plasma leucine concentrations were lower in the inversion compared with the control treatment (177 ± 15 vs. 236 ± 15 mmol·L−1, p < 0.05, which was accompanied by a lower plasma essential amino acid (EAA response over 240 min (31,956 ± 6441 vs. 50,351 ± 4015 AU; p < 0.05. Peak plasma paracetamol concentrations were lower in the inversion vs. control treatment (5.8 ± 1.1 vs. 10.0 ± 0.6 mg·L−1, p < 0.05. Gastric emptying rate and post-prandial plasma amino acid availability are significantly decreased after protein ingestion in a head-down tilted position. Therefore, upright body positioning should be considered when aiming to augment post-prandial muscle protein accretion in both health and disease.

  19. Influence of gallic and tannic acids on enzymatic activity and growth ...

    African Journals Online (AJOL)

    SERVER

    2008-02-19

    Feb 19, 2008 ... The effect of phenolic acids (gallic and tannic acids) on growth of Pectobacterium chrysanhemi, and its protease and ... effect of the tannic and gallic acids on the growth of this strain. The growth rate .... antifungal products.

  20. Defective thrombus formation in mice lacking endogenous factor VII activating protease (FSAP).

    Science.gov (United States)

    Subramaniam, Saravanan; Thielmann, Ina; Morowski, Martina; Pragst, Ingo; Sandset, Per Morten; Nieswandt, Bernhard; Etscheid, Michael; Kanse, Sandip M

    2015-04-01

    Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (pendogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

  1. Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

    Science.gov (United States)

    Slijepcevic, Davor; Roscam Abbing, Reinout L P; Katafuchi, Takeshi; Blank, Antje; Donkers, Joanne M; van Hoppe, Stéphanie; de Waart, Dirk R; Tolenaars, Dagmar; van der Meer, Jonathan H M; Wildenberg, Manon; Beuers, Ulrich; Oude Elferink, Ronald P J; Schinkel, Alfred H; van de Graaf, Stan F J

    2017-11-01

    The Na + -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

  2. Luminometric method for screening retroviral protease inhibitors

    Czech Academy of Sciences Publication Activity Database

    Horáková, D.; Rumlová, Michaela; Pichová, Iva; Ruml, Tomáš

    2005-01-01

    Roč. 345, č. 1 (2005), s. 96-101 ISSN 0003-2697 R&D Projects: GA AV ČR(CZ) IAA4055304; GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : retroviral protease * inhibitors * luminescent assay Subject RIV: CE - Biochemistry Impact factor: 2.670, year: 2005

  3. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

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    Madhusudhan Budatha

    Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

  4. Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots.

    Science.gov (United States)

    Plett, Jonathan M; Khachane, Amit; Ouassou, Malika; Sundberg, Björn; Kohler, Annegret; Martin, Francis

    2014-04-01

    The plant hormones ethylene, jasmonic acid and salicylic acid have interconnecting roles during the response of plant tissues to mutualistic and pathogenic symbionts. We used morphological studies of transgenic- or hormone-treated Populus roots as well as whole-genome oligoarrays to examine how these hormones affect root colonization by the mutualistic ectomycorrhizal fungus Laccaria bicolor S238N. We found that genes regulated by ethylene, jasmonic acid and salicylic acid were regulated in the late stages of the interaction between L. bicolor and poplar. Both ethylene and jasmonic acid treatments were found to impede fungal colonization of roots, and this effect was correlated to an increase in the expression of certain transcription factors (e.g. ETHYLENE RESPONSE FACTOR1) and a decrease in the expression of genes associated with microbial perception and cell wall modification. Further, we found that ethylene and jasmonic acid showed extensive transcriptional cross-talk, cross-talk that was opposed by salicylic acid signaling. We conclude that ethylene and jasmonic acid pathways are induced late in the colonization of root tissues in order to limit fungal growth within roots. This induction is probably an adaptive response by the plant such that its growth and vigor are not compromised by the fungus. © 2013 The Authors New Phytologist © 2013 New Phytologist Trust.

  5. Extracellular loop 2 of the free Fatty Acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator

    DEFF Research Database (Denmark)

    Smith, Nicola J; Ward, Richard J; Stoddart, Leigh A

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molec...

  6. Nitro-oleic acid modulates classical and regulatory activation of macrophages and their involvement in pro-fibrotic responses

    Czech Academy of Sciences Publication Activity Database

    Ambrožová, Gabriela; Martíšková, Hana; Koudelka, A.; Ravekes, T.; Rudolph, T.K.; Klinke, A.; Rudolph, V.; Freeman, B.A.; Woodcock, S.R.; Kubala, Lukáš; Pekarová, Michaela

    2016-01-01

    Roč. 90, JAN 2016 (2016), s. 252-260 ISSN 0891-5849 R&D Projects: GA ČR(CZ) GP13-40824P Grant - others:GAAV(CZ) M200041208 Institutional support: RVO:68081707 Keywords : Nitro-fatty acids * Nitro-oleic acid * Macrophages Subject RIV: BO - Biophysics Impact factor: 5.606, year: 2016

  7. Role of Proteases in Chronic Obstructive Pulmonary Disease

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    Kailash C. Pandey

    2017-08-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

  8. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

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    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  9. Corruption of innate immunity by bacterial proteases.

    Science.gov (United States)

    Potempa, Jan; Pike, Robert N

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host's innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections.

  10. Changes in IgA protease expression are conferred by changes in genomes during persistent infection by nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Gallo, Mary C; Kirkham, Charmaine; Eng, Samantha; Bebawee, Remon S; Kong, Yong; Pettigrew, Melinda M; Tettelin, Hervé; Murphy, Timothy F

    2018-05-14

    Nontypeable Haemophilus influenzae (NTHi) is an exclusively human pathobiont that plays a critical role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD). NTHi causes acute exacerbations of COPD and also causes persistent infection of the lower airways. NTHi expresses four IgA protease variants (A1, A2, B1, and B2) that play different roles in virulence. Expression of IgA proteases varies among NTHi strains, but little is known about the frequency and mechanisms by which NTHi modulates IgA protease expression during infection in COPD. To assess expression of IgA protease during natural infection in COPD, we studied IgA protease expression of 101 persistent strains (median duration of persistence 161 days, range 2 to 1422) collected longitudinally from patients enrolled in a 20-year study of COPD upon initial acquisition and immediately before clearance from the host. Upon acquisition, 89 (88%) expressed IgA protease. A total of 16 of 101 (16%) strains of NTHi altered expression of IgA protease during persistence. Indels and slipped-strand mispairing of mononucleotide repeats conferred changes in expression of igaA1, igaA2, and igaB1 Strains with igaB2 underwent frequent changes in expression of IgA protease B2 during persistence, mediated by slipped-strand mispairing of a 7-nucleotide repeat, TCAAAAT, within the open reading frame of igaB2 We conclude that changes in iga gene sequences result in changes in expression of IgA proteases by NTHi during persistent infection in the respiratory tract of patients with COPD. Copyright © 2018 American Society for Microbiology.

  11. Substrate modulation of fatty acid effects on energization and respiration of kidney proximal tubules during hypoxia/reoxygenation.

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    Anja Bienholz

    Full Text Available Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.

  12. Improved Protease-Targeting and Biopharmaceutical Properties of Novel Prodrugs of Ganciclovir.

    Science.gov (United States)

    Sun, Kefeng; Xu, Hao; Hilfinger, John L; Lee, Kyung-Dall; Provoda, Chester J; Sabit, Hairat; Amidon, Gordon L

    2018-02-05

    The prodrug strategy has been frequently employed as a chemical approach for overcoming the disadvantages of existing parent drugs. In this report, we synthesized four monoester prodrugs of ganciclovir, an anticytomegalovirus drug, and demonstrated their potential advantages in protease-targeted activation and biopharmaceutical profiles over the parent compound. We demonstrated that these four prodrugs of ganciclovir, i.e., N-benzyloxycarbonyl-(L)-alanine-ganciclovir (CbzAlaGCV), N-benzyloxycarbonyl-(α,l)-aminobutyric acid-ganciclovir (CbzAbuGCV), N-acetyl-(l)-phenylalanine-(l)-alanine-ganciclovir (AcPheAlaGCV), and N-acetyl-(l)-phenylalanine-(α,l)-aminobutyric acid-ganciclovir (AcPheAbuGCV), are hydrolytically activated by the protease of human cytomegalovirus (hCMV), a serine protease that possesses intrinsic esterase activities. CbzAlaGCV and AcPheAlaGCV were found to be activated at a higher rate by the hCMV protease than CbzAbuGCV and AcPheAbuGCV. These ganciclovir prodrugs could potentially be targeted to selective activation by the hCMV protease which is only present at the viral infection sites, thereby achieving higher efficacy and lower systemic toxicity. The tissue stability, cellular uptake, and trans-epithelial transport of these ganciclovir prodrugs were also characterized. The N-acetylated dipeptide prodrugs of ganciclovir were found to be generally more stable than Cbz-amino acid prodrugs in various tissue matrices. Among the four prodrug candidates, AcPheAbuGCV was the most stable in human cell homogenates, plasma, and pooled liver microsomes. AcPheAbuGCV also possessed a superior cellular uptake profile and permeability across epithelial cell monolayers. Since the targeting and selective activation of a prodrug is determined by not only its rate of hydrolysis catalyzed by the hCMV protease target but also its biopharmaceutical properties, i.e., oral absorption and systemic availability, AcPheAbuGCV is considered the best overall candidate among

  13. Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

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    Porntip Pinlaor

    Full Text Available The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and

  14. A novel ascorbic acid sensor based on the Fe3+/Fe2+ modulated photoluminescence of CdTe quantum dots@SiO2 nanobeads.

    Science.gov (United States)

    Ma, Qiang; Li, Yang; Lin, Zi-Han; Tang, Guangchao; Su, Xing-Guang

    2013-10-21

    In this paper, CdTe quantum dot (QD)@silica nanobeads were used as modulated photoluminescence (PL) sensors for the sensing of ascorbic acid in aqueous solution for the first time. The sensor was developed based on the different quenching effects of Fe(2+) and Fe(3+) on the PL intensity of the CdTe QD@ silica nanobeads. Firstly, the PL intensity of the CdTe QDs was quenched in the presence of Fe(3+). Although both Fe(2+) and Fe(3+) could quench the PL intensity of the CdTe QDs, the quenching efficiency were quite different for Fe(2+) and Fe(3+). The PL intensity of the CdTe QD@silica nanobeads can be quenched by about 15% after the addition of Fe(3+) (60 μmol L(-1)), while the PL intensity of the CdTe QD@silica nanobeads can be quenched about 49% after the addition of Fe(2+) (60 μmol L(-1)). Therefore, the PL intensity of the CdTe QD@silica nanobeads decreased significantly when Fe(3+) was reduced to Fe(2+) by ascorbic acid. To confirm the strategy of PL modulation in this sensing system, trace H2O2 was introduced to oxidize Fe(2+) to Fe(3+). As a result, the PL intensity of the CdTe QD@silica nanobeads was partly recovered. The proposed sensor could be used for ascorbic acid sensing in the concentration range of 3.33-400 μmol L(-1), with a detection limit (3σ) of 1.25 μmol L(-1) The feasibility of the proposed sensor for ascorbic acid determination in tablet samples was also studied, and satisfactory results were obtained.

  15. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

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    Rachana Fulzele

    2011-12-01

    Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

  16. Alpha-fetoprotein (AFP) modulates the effect of serum albumin on brain development by restraining the neurotrophic effect of oleic acid.

    Science.gov (United States)

    García-García, Alejandro G; Polo-Hernández, Erica; Tabernero, Arantxa; Medina, José M

    2015-10-22

    We have previously shown that serum albumin controls perinatal rat brain development through the regulation of oleic acid synthesis by astrocytes. In fact, oleic acid synthesized and released by astrocytes promoted neurite growth, neuron migration and the arrangement of prospective synapses. In this work we show that alpha-fetoprotein (AFP) is also present in the brain during embryonic development, its concentrations peaking at E15.5 and at E19.5. However, after E19.5 AFP concentrations plummeted concurrently with a sharp increase in serum albumin concentrations. At E15.5, AFP is present in caudal regions of the brain, particularly in brain areas undergoing differentiation during this period, such as the thalamic reticular nucleus of the thalamus, the hypothalamus, the amygdala and the hippocampus. Albumin was not detected in the brain at E15.5 but stained brain cells substantially on day E19.5, showing a very similar distribution to that of AFP under the same circumstances. The concentrations of free oleic acid in the brain were inversely correlated with those of AFP, suggesting that the signals elicited by AFP and oleic acid can be inversely associated. GAP-43, a marker of axonal growth that is highly expressed by the presence of oleic acid, was not co-localized with AFP except in the marginal zone and areas delimiting the subplate. AFP prevented the increase in GAP-43 expression caused by the presence of oleic acid in neurons in primary culture in vitro and in organotypic cultures of embryonic rat brain ex vivo, suggesting that AFP may modulate the effect of serum albumin on brain development. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Cross-Sectional Imaging of Boundary Lubrication Layer Formed by Fatty Acid by Means of Frequency-Modulation Atomic Force Microscopy.

    Science.gov (United States)

    Hirayama, Tomoko; Kawamura, Ryota; Fujino, Keita; Matsuoka, Takashi; Komiya, Hiroshi; Onishi, Hiroshi

    2017-10-10

    To observe in situ the adsorption of fatty acid onto metal surfaces, cross-sectional images of the adsorption layer were acquired by frequency-modulation atomic force microscopy (FM-AFM). Hexadecane and palmitic acid were used as the base oil and typical fatty acid, respectively. A Cu-coated silicon wafer was prepared as the target substrate. The solvation structure formed by hexadecane molecules at the interface between the Cu substrate and the hexadecane was observed, and the layer pitch was found to be about 0.6 nm, which corresponds to the height of hexadecane molecules. This demonstrates that hexadecane molecules physically adsorbed onto the surface due to van der Waals forces with lying orientation because hexadecane is a nonpolar hydrocarbon. When hexadecane with palmitic acid was put on the Cu substrate instead of pure hexadecane, an adsorption layer of palmitic acid was observed at the interface. The layer pitch was about 2.5-2.8 nm, which matches the chain length of palmitic acid molecules well. This indicates that the original adsorption layer was monolayer or single bilayer in the local area. In addition, a cross-sectional image captured 1 h after observation started to reveal that the adsorbed additive layer gradually grew up to be thicker than about 20 nm due to an external stimulus, such as cantilever oscillation. This is the first report of in situ observation of an adsorbed layer by FM-AFM in the tribology field and demonstrates that FM-AFM is useful for clarifying the actual boundary lubrication mechanism.

  18. Towards tricking a pathogen's protease into fighting infection: the 3D structure of a stable circularly permuted onconase variant cleavedby HIV-1 protease.

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    Mariona Callís

    Full Text Available Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease's activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.

  19. Excitatory amino acid transporters: recent insights into molecular mechanisms, novel modes of modulation and new therapeutic possibilities

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Fahlke, Christoph; Bjørn-Yoshimoto, Walden Emil

    2015-01-01

    The five excitatory amino acid transporters (EAAT1–5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations into the ......The five excitatory amino acid transporters (EAAT1–5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations...

  20. Arabidopsis Glutamate Receptor Homolog3.5 Modulates Cytosolic Ca2+ Level to Counteract Effect of Abscisic Acid in Seed Germination1[OPEN

    Science.gov (United States)

    Kong, Dongdong; Ju, Chuanli; Parihar, Aisha; Kim, So; Cho, Daeshik; Kwak, June M.

    2015-01-01

    Seed germination is a critical step in a plant’s life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. However, the molecular mechanisms underlying germination control remain elusive. Here, we report that the Arabidopsis (Arabidopsis thaliana) glutamate receptor homolog3.5 (AtGLR3.5) is predominantly expressed in germinating seeds and increases cytosolic Ca2+ concentration that counteracts the effect of abscisic acid (ABA) to promote germination. Repression of AtGLR3.5 impairs cytosolic Ca2+ concentration elevation, significantly delays germination, and enhances ABA sensitivity in seeds, whereas overexpression of AtGLR3.5 results in earlier germination and reduced seed sensitivity to ABA. Furthermore, we show that Ca2+ suppresses the expression of ABSCISIC ACID INSENSITIVE4 (ABI4), a key transcription factor involved in ABA response in seeds, and that ABI4 plays a fundamental role in modulation of Ca2+-dependent germination. Taken together, our results provide molecular genetic evidence that AtGLR3.5-mediated Ca2+ influx stimulates seed germination by antagonizing the inhibitory effects of ABA through suppression of ABI4. These findings establish, to our knowledge, a new and pivotal role of the plant glutamate receptor homolog and Ca2+ signaling in germination control and uncover the orchestrated modulation of the AtGLR3.5-mediated Ca2+ signal and ABA signaling via ABI4 to fine-tune the crucial developmental process, germination, in Arabidopsis. PMID:25681329

  1. A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

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    Joseph E Ippolito

    Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  2. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxybenzoic Acid

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    Kuan-Han Lee

    2014-01-01

    Full Text Available Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxybenzoic acid (TMPBA and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 µM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4',6-diamidino-2-phenylindole (DAPI nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP kinases, 5' adenosine monophosphate-activated protein kinase (AMPK, and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

  3. Erwinia carotovora extracellular proteases : characterization and role in soft rot

    OpenAIRE

    Kyöstiö, Sirkka R. M.

    1990-01-01

    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

  4. Economic Methods of Ginger Protease'sextraction and Purification

    Science.gov (United States)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  5. Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

    International Nuclear Information System (INIS)

    Wells, P.G.; Zubovits, J.T.; Wong, S.T.; Molinari, L.M.; Ali, S.

    1989-01-01

    Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeic acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05)

  6. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.

    Directory of Open Access Journals (Sweden)

    André N Mueller

    2013-02-01

    Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi.

  7. Structural Insights of the Cysteine Protease Heynein from Induction and Characterization of Non-native Intermediate States

    Directory of Open Access Journals (Sweden)

    Basant K. Patel

    2010-12-01

    Full Text Available Cysteine proteases are vital to cell physiology and many plants secrete these proteases for defense purposes. Many recent studies have reported unusually high stabilities for several plant cysteine proteases which possibly enable these proteases to function under adverse environmental conditions. Here, we have examined the conformational features of a new plant cysteine protease heynein using spectroscopic tools to understand the basis for its robust functional stability. The studies revealed structural integrity over a wide range of pH (2.5-12.0, temperature (65 oC and urea (8M. However, at pH 2.0, the protein gets acid-unfolded (UA -state with exposed hydrophobic patches, which upon addition of more protons (pH 0.5 or anions (0.5 M KCl and 0.2 M Na2 SO4 yields conformationally distinct refolded intermediates respectively termed: A-, I 1 - and I 2 -states. Strikingly, a high methanol level drives the UA -state into a predominantly -sheet rich conformation (O-state. We observed three-state unfolding kinetics of the I 2 -state by urea, possibly suggesting presence of two domains in the heynein molecule.

  8. Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

    Science.gov (United States)

    Chatterjee, Joyee; Giri, Sudipta; Maity, Sujan; Sinha, Ankan; Ranjan, Ashish; Rajshekhar; Gupta, Suvroma

    2015-01-01

    Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  9. [Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

    Science.gov (United States)

    Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

    1997-01-01

    Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

  10. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and w......Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

  11. Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

    Directory of Open Access Journals (Sweden)

    Tanveer Majeed

    2013-01-01

    Full Text Available A putative protease gene (aprE from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10. In addition, the enzyme had an elevated optimum temperature (60°C. The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

  12. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Directory of Open Access Journals (Sweden)

    Suk Ho Choi

    2011-09-01

    Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

  13. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  14. Maternal and neonatal dietary intake of balanced n-6/n-3 fatty acids modulates experimental colitis in young adult rats.

    Science.gov (United States)

    Reddy, K Vijay Kumar; Naidu, K Akhilender

    2016-08-01

    The imbalance of n-6 and n-3 polyunsaturated fatty acids in the maternal diet impairs intestinal barrier development and sensitizes the colon response to inflammatory insults in the young rats. With a view to overcoming this issue, we designed this study to investigate the effect of maternal and neonatal intake of different proportions of n-6/n-3 fatty acids on colon inflammation in the young adult rats. Female Wistar rats were assigned into four groups, and each group fed one of four semisynthetic diets, namely n-6, low n-3, n-6/n-3 and n-3 fatty acids for 8 weeks prior to mating, during gestation and lactation periods. At weaning, the pups were separated from the dams and fed diet similar to the mothers. Colitis was induced on postnatal day 35, by administering 2 % dextran sulfate sodium in drinking water for 10 days. Colitis was assessed based on the clinical and inflammatory markers in the colon. Fatty acid analysis was done in liver, RBC, colon and spleen. A balanced n-6/n-3 PUFA diet significantly improved the body weight loss, rectal bleeding and mortality in rats. This was associated with lower myeloperoxidase activity, nitric oxide, prostaglandin E2, TNF-α and IL-6, IL-8, COX-2 and iNOS levels in the colon tissues. Fatty acid analysis has shown that the arachidonic acid/docosahexaenoic acid ratio was significantly lower in liver, RBC, colon and spleen in n-6/n-3 and n-3 diet groups. We demonstrate that balanced n-6/n-3 PUFA supplementation in maternal and neonatal diet alters systemic AA/DHA ratio and attenuates colon inflammation in the young adult rats.

  15. The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition.

    Science.gov (United States)

    Casilag, Fiordiligie; Lorenz, Anne; Krueger, Jonas; Klawonn, Frank; Weiss, Siegfried; Häussler, Susanne

    2016-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

    2002-08-01

    In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

  17. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  18. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

    Science.gov (United States)

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

    2016-06-01

    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

    Science.gov (United States)

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Pandian, Shunmugiah Karutha

    2011-10-01

    Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

  20. Dual orexin receptor antagonists show distinct effects on locomotor performance, ethanol interaction and sleep architecture relative to gamma-aminobutyric acid-A receptor modulators

    Directory of Open Access Journals (Sweden)

    Andres D. Ramirez

    2013-12-01

    Full Text Available Dual orexin receptor antagonists (DORAs are a potential treatment for insomnia that function by blocking both the orexin 1 and orexin 2 receptors. The objective of the current study was to further confirm the impact of therapeutic mechanisms targeting insomnia on locomotor coordination and ethanol interaction using DORAs and gamma-aminobutyric acid (GABA-A receptor modulators of distinct chemical structure and pharmacologic properties in the context of sleep-promoting potential. The current study compared rat motor co-ordination after administration of DORAs, DORA-12 and almorexant, and GABA-A receptor modulators, zolpidem, eszopiclone and diazepam, alone or each in combination with ethanol. Motor performance was assessed by measuring time spent walking on a rotarod apparatus. Zolpidem, eszopiclone and diazepam (0.3–30 mg/kg administered orally [PO] impaired rotarod performance in a dose-dependent manner. Furthermore, all three GABA-A receptor modulators potentiated ethanol- (0.25–1.25 g/kg induced impairment on the rotarod. By contrast, neither DORA-12 (10–100 mg/kg, PO nor almorexant (30–300 mg/kg, PO impaired motor performance alone or in combination with ethanol. In addition, distinct differences in sleep architecture were observed between ethanol, GABA-A receptor modulators (zolpidem, eszopiclone and diazepam and DORA-12 in electroencephalogram studies in rats. These findings provide further evidence that orexin receptor antagonists have an improved motor side-effect profile compared with currently available sleep-promoting agents based on preclinical data and strengthen the rationale for further evaluation of these agents in clinical development.

  1. Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

    OpenAIRE

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A.; Saul, Allan; Gerke, Christiane

    2014-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Int...

  2. Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio: evidence for subfunctionalization or neofunctionalization of duplicated genes

    Directory of Open Access Journals (Sweden)

    Denovan-Wright Eileen M

    2009-09-01

    Full Text Available Abstract Background In the Duplication-Degeneration-Complementation (DDC model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps genes by dietary fatty acids (FAs in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid, sunflower oil (12% lipid, rich in linoleic acid, linseed oil (12% lipid, rich in linolenic acid, or low fat (4% lipid, low fat diet for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. Result FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR. In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases

  3. TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

    Science.gov (United States)

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

    2016-12-15

    The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope

  4. Effect of Microenvironmental pH Modulation on the Dissolution Rate and Oral Absorption of the Salt of a Weak Acid - Case Study of GDC-0810.

    Science.gov (United States)

    Hou, Hao Helen; Jia, Wei; Liu, Lichuan; Cheeti, Sravanthi; Li, Jane; Nauka, Ewa; Nagapudi, Karthik

    2018-01-29

    The purpose of this work is to investigate the effect of microenvironmental pH modulation on the in vitro dissolution rate and oral absorption of GDC-0810, an oral anti-cancer drug, in human. The pH-solubility profile of GDC-0810 free acid and pH max of its N-Methyl-D-glucamine (NMG) salt were determined. Precipitation studies were conducted for GDC-0810 NMG salt at different pH values. GDC-0810 200-mg dose NMG salt tablet formulations containing different levels of sodium bicarbonate as the pH modifier were tested for dissolution under the dual pH-dilution scheme. Three tablet formulations were evaluated in human as a part of a relative bioavailability study. A 200-mg dose of GDC-0810 was administered QD with low fat food. Intrinsic solubility of GDC-0810 free acid was found to be extremely low. The pH max of the NMG salt suggested a strong tendency for form conversion to the free acid under GI conditions. In vitro dissolution profiles showed that the dissolution rate and extent of GDC-0810 increased with increasing the level of sodium bicarbonate in the formulation. The human PK data showed a similar trend for the geometric mean of C max and AUC 0-t for formulations containing 5%, 10%, and 15% sodium bicarbonate, but the difference is not statistically significant. Incorporation of a basic pH modifier, sodium bicarbonate, in GDC-0810 NMG salt tablet formulations enhanced in vitro dissolution rate of GDC-0810 via microenvironmental pH modulation. The human PK data showed no statistically significant difference in drug exposure from tablets containing 5%, 10%, and 15% sodium bicarbonate.

  5. Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate.

    Science.gov (United States)

    Pillai, Priya; Archana, G

    2008-03-01

    Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45-50 degrees C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65 degrees C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.

  6. Milk fatty acid profile is modulated by DGAT1 and SCD1 genotypes in dairy cattle on pasture and strategic supplementation.

    Science.gov (United States)

    Carvajal, A M; Huircan, P; Dezamour, J M; Subiabre, I; Kerr, B; Morales, R; Ungerfeld, E M

    2016-05-09

    Milk fat composition is important to consumer health. During the last decade, some fatty acids (FA) have received attention because of their functional and beneficial effects on human health. The milk FA profile is affected by both diet and genetics. Differences in milk fat composition are based on biochemical pathways, and candidate genes have been proposed to explain FA profile variation. Here, the association between DGAT1 K232A, SCD1 A293V, and LEPR T945M markers with milk fat composition in southern Chile was evaluated. We selected five herds of Holstein-Friesian, Jersey, Frisón Negro, Montbeliarde, and Overo Colorado cows (pasture-grazed) that received strategic supplementation with concentrates and conserved forages. We genotyped the SNPs and calculated allele frequencies and Hardy-Weinberg equilibrium. Milk fat composition was determined for individual milk samples over a year, and associations between genotypes and milk composition were studied. The most frequent variants for DGAT1, SCD1, and LEPR polymorphisms were GC/GC, C, and C, respectively. The DGAT1 GC/GC allele was associated with lower milk fat and protein content, lower saturated fatty acid levels, and higher polyunsaturated FA (PUFA), n-3 and n-6 FA, and a linolenic acid to cholesterolemic FA ratios, which implied a healthier FA profile. The SCD1 CC genotype was associated with a low cholesterolemic FA content, a high ratio of linolenic acid to cholesterolemic FA, and lower conjugated-linolenic acid and PUFA content. These results suggest the possible modulation of milk fat profiles, using specific genotypes, to improve the nutritional quality of dairy products.

  7. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Long chain polyunsaturated fatty acids (LCPUFAs) and nordihydroguaiaretic acid (NDGA) modulate metabolic and inflammatory markers in a spontaneous type 2 diabetes mellitus model (Stillman Salgado rats).

    Science.gov (United States)

    Dain, Alejandro; Repossi, Gaston; Diaz-Gerevini, Gustavo T; Vanamala, Jairam; Das, Undurti N; Eynard, Aldo R

    2016-11-25

    Diabetes mellitus (DM) is a complex disease with alterations in metabolic and inflammatory markers. Stillman Salgado rats (eSS) spontaneously develop type 2 DM by middle age showing progressive impairment of glucose tolerance with hyperglycemia, hypertriglyceridemia and hyperinsulinemia. We analyzed the effects of supplementation of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) with or without nordihydroguaiaretic acid (NDGA) added, an antioxidant and lipoxygenase inhibitor, on metabolic and inflammatory parameters in eSS rats to evaluate whether they can delay development and/or prevent progression of DM. After weaning, eSS rats received, intraperitoneally, once a month ω-3 (EPA 35% and DHA 40%-6.25 mg/Kg) or ω-6 (90% arachidonic acid- 6. 25 mg/Kg) for twelve months. Two additional groups of rats received 1.9 mg/kg NDGA added to ω-3 and ω-6 fatty acids. Blood samples were collected at day 40, and at the end of the 6th month and 12th month of age to determine plasma triglycerides (TGs), total plasma fatty acids (FA), A1C hemoglobin (HbA1C), C-reactive protein (CRP), gamma glutamyl transpeptidase (GGT), lipo and hydro peroxides, nitrites and IL-6 (in plasma and liver, kidney, and pancreas) and underwent oral glucose tolerance test (OGTT) as well. Wistar and eSS rats that received saline solution were used as controls. Plasma lipids profile, TG, fasting and post-prandial blood glucose levels, and glycosylated HbA1C showed significant improvements in ω-3 and ω-3 + NDGA treated animals compared to eSS control group. ω-3 and ω-3 + NDGA groups showed an inverse correlation with fasting blood glucose and showed lower plasma levels of GGT, TG, and CRP. eSS rats treated with ω-3 LCPUFAs showed reduced level of inflammatory and oxidative indices in plasma and liver, kidney and pancreas tissues in comparison with eSS control (non-treated) and ω-6 treated groups. eSS rats are a useful model to study type 2 DM pathophysiology and related inflammatory

  9. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Energy Technology Data Exchange (ETDEWEB)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  10. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    International Nuclear Information System (INIS)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group

  11. Characte