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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

    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...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  2. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    -specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers......, 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...

  3. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A. (UMASS, MED)

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  4. Acid protease production in fungal root endophytes.

    Science.gov (United States)

    Mayerhofer, Michael S; Fraser, Erica; Kernaghan, Gavin

    2015-01-01

    Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... Proteases with high activity and stability in acid pH range have .... at a flow rate of 25 ml/h after elution with the same buffer. Protein content (abs. at ..... Role of the hydrophobic amino acid residue in the bitterness of peptides.

  6. Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

    Institute of Scientific and Technical Information of China (English)

    LI Jing; PENG Ying; WANG Xianghong; CHI Zhenming

    2010-01-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose,1.5% casein,and 0.5% yeast extract,and the optimal cultivation conditions of the acid protease production were pH 4.0,a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions,72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese,food and fermentation industries.

  7. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    Science.gov (United States)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  8. Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana.

    Science.gov (United States)

    Bailey, Mark; Srivastava, Anjil; Conti, Lucio; Nelis, Stuart; Zhang, Cunjin; Florance, Hannah; Love, Andrew; Milner, Joel; Napier, Richard; Grant, Murray; Sadanandom, Ari

    2016-01-01

    Small ubiquitin-like modifier proteases 1 and 2 (SUMO1/2) have been linked to the regulation of salicylic acid (SA)-mediated defence signalling in Arabidopsis thaliana. In order to define the role of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS1/2) in defence and to provide insight into SUMO1/2-mediated regulation of SA signalling, we examined the status of SA-mediated defences in ots1/2 mutants. The ots1 ots2 double mutant displayed enhanced resistance to virulent Pseudomonas syringae and higher levels of SA compared with wild-type (WT) plants. Furthermore, ots1 ots2 mutants exhibited upregulated expression of the SA biosynthesis gene ICS1 in addition to enhanced SA-responsive ICS1 expression beyond that of WT. SA stimulated OTS1/2 degradation and promoted accumulation of SUMO1/2 conjugates. These results indicate that OTS1 and -2 act in a feedback loop in SA signalling and that de novo OTS1/2 synthesis works antagonistically to SA-promoted degradation, adjusting the abundance of OTS1/2 to moderate SA signalling. Accumulation of SUMO1/2 conjugates coincides with SA-promoted OTS degradation and may play a positive role in SA-mediated signalling in addition to its repressive roles reported elsewhere.

  9. 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

    2015-12-01

    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.

  10. Flexible catalytic site conformations implicated in modulation of HIV-1 protease autoprocessing reactions

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2011-10-01

    Full Text Available Abstract Background The HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell. Protease autoprocessing, by which the protease domain embedded in the precursor catalyzes essential cleavage reactions, leads to liberation of the free mature protease at the late stage of the replication cycle. To examine autoprocessing reactions in transfected mammalian cells, we previously described an assay using a fusion precursor consisting of the mature protease (PR along with its upstream transframe region (p6* sandwiched between GST and a small peptide epitope. Results In this report, we studied two autoprocessing cleavage reactions, one between p6* and PR (the proximal site and the other in the N-terminal region of p6* (the distal site catalyzed by the embedded protease, using our cell-based assay. A fusion precursor carrying the NL4-3 derived protease cleaved both sites, whereas a precursor with a pseudo wild type protease preferentially autoprocessed the proximal site. Mutagenesis analysis demonstrated that several residues outside the active site (Q7, L33, N37, L63, C67 and H69 contributed to the differential substrate specificity. Furthermore, the cleavage reaction at the proximal site mediated by the embedded protease in precursors carrying different protease sequences or C-terminal fusion peptides displayed varied sensitivity to inhibition by darunavir, a catalytic site inhibitor. On the other hand, polypeptides such as a GCN4 motif, GFP, or hsp70 fused to the N-terminus of p6* had a minimal effect on darunavir inhibition of either cleavage reaction. Conclusions Taken together, our data suggest that several non-active site residues and the C-terminal flanking peptides regulate embedded protease activity through modulation of the catalytic site conformation. The cell-based assay provides a sensitive tool to study protease autoprocessing reactions in mammalian cells.

  11. 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.

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

    Science.gov (United States)

    Butterworth, Michael B; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M; Thibodeau, Patrick H

    2014-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases.

    Science.gov (United States)

    Oka, Tomoichiro; Murakami, Kosuke; Wakita, Takaji; Katayama, Kazuhiko

    2011-02-01

    Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino-acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  15. Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen

    Institute of Scientific and Technical Information of China (English)

    Zeng-Jun Wang; Wei Zhang; Ning-Han Feng; Ning-Hong Song; Hong-Fei Wu; Yuan-Geng Sui

    2008-01-01

    Aim: To study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg). Methods: Human Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-423) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored. Results: Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin. Conclusion: Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

  16. Scouring Potential of Mesophile Acidic Proteases of Pseudomonas aeruginosa for Grey Cotton Fabrics

    Science.gov (United States)

    Saravanan, D.

    2013-04-01

    Mesophile, acidic proteases were produced using the microbial source, Pseudomonas aeruginosa, with wider thermal tolerances. Process conditions of scouring treatment were optimized using Taguchi method for optimum temperature, time, pH and concentration of protease. Treatment with the protease lower weight loss values compared to the alkali scouring, however, significant improvement in the absorbency compared to the grey samples was observed. Large amounts of pectin left out in the samples resulted in higher extractable impurities, substantiated by the FTIR results. Relatively, lower reduction in the tear strengths was observed in both warp and weft directions after protease treatment of the cotton fabrics.

  17. 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.

  18. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Science.gov (United States)

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  19. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Directory of Open Access Journals (Sweden)

    Adela Rendón-Ramírez

    Full Text Available Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP. CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP and a scaffold recognizing a β-lactam (imipenem in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV, we sought to query other OMV proteins, like phospholipase C (PLC, using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM. Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  20. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    Energy Technology Data Exchange (ETDEWEB)

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George; (SPRI)

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  1. HIV Protease Inhibitor-Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation.

    Science.gov (United States)

    Kourjian, Georgio; Rucevic, Marijana; Berberich, Matthew J; Dinter, Jens; Wambua, Daniel; Boucau, Julie; Le Gall, Sylvie

    2016-05-01

    Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.

  2. Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores of Bacillus Species Is an Atypical Aspartic Acid Protease

    Science.gov (United States)

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

    Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582

  3. Methods for producing dairy products, in particular cheese using lactic acid bacteria provided with additional neutral protease activity

    NARCIS (Netherlands)

    Mierau, Igor; van den Burg, Lambertus; Kok, Jan; Venema, Gerhardus

    1997-01-01

    The present invention relates to a method for carrying out a process of biotransformation of a substrate whereby at least one lactic acid bacterium comprising a gene encoding a neutral protease having an altered resulting activity, e.g. a neutral protease having an altered stability and/or specifici

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

    Science.gov (United States)

    Monnerie, Hubert; Le Roux, Peter D

    2008-09-01

    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    Science.gov (United States)

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  8. 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

  9. Supplementation of corn-soybean based layer diets with different levels of acid protease

    Directory of Open Access Journals (Sweden)

    Satrijo Widi Purbojo

    2004-03-01

    Full Text Available The aim of this research was held in Institute of Animal Science farm, University of the Philippines Los Banos, was to know the effects of acid protease supplementation in layer diets. Ninety-four-week old pullets were caged individually. Five treatments were randomly arranged to 95 pullets following a completely randomized design. Each treatment was replicated 19 times. The feeding trial lasted for 16 weeks. The same management practices were provided to all treatments throughout the feeding period. Pullets were fed once a day in the morning and clean drinking water was available to the pullets at all times. A basal layer diet that contained 18% crude protein (CP and 2800 Kcal ME/kg supplemented with required vitamins, minerals and amino acids were formulated. The diets with reduced protein of 17% and 2800 kcal ME/kg was also formulated. The diet with the reduced crude protein was supplemented with different levels of protease (0.05, 0.075 and 0.1%. Six birds from each treatment were randomly selected and placed in individual digestion cages. They were fed with their respective diets with chromic oxide as indicator for 7 days. Chromic oxide was added to the different diets at 0.2%. On the 3rd to 6th day of feeding, feces were collected using stainless fecal trays installed under each cage. At the end of the collection period, fecal samples collected from each replicate of treatment were dried then subjected to proximate analysis and chromic oxide determination. Result showed that no significant difference on biweekly feed consumption and overall observations. Reduced CP + 0.1% protease was the highest on the hen day production (93.75% and the lowest of feed conversion (1.85 while reduced CP + 0.075% protease was the highest of egg weight (58.82 g and eggshell thickness (0.392 mm. There was no significant difference on digestibility coefficient.

  10. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

    Science.gov (United States)

    Fung, G; Shi, J; Deng, H; Hou, J; Wang, C; Hong, A; Zhang, J; Jia, W; Luo, H

    2015-01-01

    We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin–protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at amino-acid 327 between glutamine and alanine, resulting in the generation of an N- and C-terminal cleavage fragment of ~35 and ~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. PMID

  11. Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors.

    Science.gov (United States)

    Zhao, Hua; Holmes, Shaletha S; Baker, Gary A; Challa, Suresh; Bose, Himangshu S; Song, Zhiyan

    2012-10-01

    Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 μg mL(-1)) was one-third that of wide-type betulinic acid (60 μg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.

  12. Pentapeptide boronic acid inhibitors of Mycobacterium tuberculosis MycP1 protease.

    Science.gov (United States)

    Frasinyuk, Mykhaylo S; Kwiatkowski, Stefan; Wagner, Jonathan M; Evans, Timothy J; Reed, Robert W; Korotkov, Konstantin V; Watt, David S

    2014-08-01

    Mycosin protease-1 (MycP1) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP1 variants from Mycobacterium thermoresistible (MycP1mth), Mycobacterium smegmatis (MycP1msm) and M. tuberculosis (MycP1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)2 (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO2(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC50 values 121.6±25.3 μM for MycP1mth, 93.2±37.3 μM for MycP1msm and 37.9±5.2 μM for MycP1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP1 catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Modulation of histamine release from human colon mast cells by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-Heng He; Hua Xie

    2004-01-01

    AIM: To investigate the ability of protease inhibitors to modulate histamine release from human colon mast cells.METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors, and histamine release was determined.RESULTS: IgE dependent histamine release from colon mast cells was inhibited by up to approximately 37%, 26% and 36.8% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-Tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, inhibitors of tryptase leupeptin, N-tosyl-L-lysine chloromethyl ketone (TLCK), lactoferrin and protamine were also able to inhibit anti-IgE induced histamine release by a maximum of some 48%, 37%, 40% and 34%, respectively. Preincubation of these inhibitors with cells for 20 min before challenged with anti-IgE had small effect on the inhibitory actions of these inhibitors on colon mast cells. A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced histamine release. The significant inhibition of calcium ionophore induced histamine release was also observed with the inhibitors of tryptase and chymase examined. Apart from leupeptin and protamine, the inhibitors tested by themselves did not stimulate colon mast cells.CONCLUSION: It was demonstrated that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced histamine release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

  14. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    HEShao-Heng; CHENPu; CHENHan-Qiu

    2003-01-01

    AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similarexperimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similarprocedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacrylgel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined byenzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native proteaseinhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain,benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsininhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymaticactivity of chymase. Native protease inhibitors α-antitrypsin and secretory leukocyte protease inhibitor (SLPI)inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymaticactivity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitorstested had relatively good selectivity to either tryptase or chymase.

  15. The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

    Science.gov (United States)

    Salgado-Pabon, Wilmara; Meyerholz, David K.; White, Mark J.; Schlievert, Patrick M.

    2016-01-01

    ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade

  16. 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.

  17. 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

    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.......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...

  18. Protease Inhibition by Oleic Acid Transfer From Chronic Wound Dressings to Albumin

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, J. V.; Howley, Phyllis; Davis, Rachel M.; Mashchak, Andrew D.; Goheen, Steven C.

    2007-08-01

    High elastase and cathepsin G activities have been observed in chronic wounds. These levels can inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid (18:1) is a non-toxic elastase inhibitor with some potential for redressing the imbalance of elastase activity found in chronic wounds. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of 18:1 by albumin under conditions mimicking chronic wounds. 18:1-treated cotton was examined for its ability to bind and release the fatty acid in the presence of albumin. The mechanism of 18:1 uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin-18:1 complexes under liquid-liquid equilibrium conditions revealed fully saturated albumin-18:1 complexes with a 1:1 weight ratio of albumin:18:1. Cotton chromatography under liquid-solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of 18:1 per mole albumin. Cotton was contrasted with hydrogel, and hydrocolloid wound dressing for its comparative ability to lower elastase activity. Each dressing material evaluated was found to release 18:1 in the presence of albumin with significant inhibition of elastase activity. The 18:1-formulated wound dressings lowered elastase activity in a dose dependent manner in the order cotton gauze > hydrogel > hydrocolloid. In contrast the cationic serine protease Cathepsin G was inihibited by 18:1 within a narrow range of 18:1-cotton formulations. Four per cent Albumin solutions were most effective in binding cotton bound-18:1. However, 2% albumin was sufficient to transfer quantities of 18:1 necessary to achieve a significant elastase-lowering effect. Formulations with 128 mg 18:1/g cotton gauze had equivalent elastase lowering with 1 - 4% albumin. 18:1 bound to cotton wound dressings may have promise in the

  19. Potential role of serine proteases in modulating renal sodium transport in vivo.

    Science.gov (United States)

    Jacquillet, G; Rubera, I; Unwin, R J

    2011-01-01

    The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.

  20. Xylanases, Cellulases, and Acid Protease Produced by Stenocarpella maydis Grown in Solid-state and Submerged Fermentation

    Directory of Open Access Journals (Sweden)

    Edna María Hernández-Domínguez

    2014-03-01

    Full Text Available Activity levels of extracellular hydrolytic enzymes produced by Stenocarpella maydis, a fungal pathogen of maize, have so far not been reported. Production of xylanase, cellulase, and acid protease by this ascomycete using different culture media in solid-state and submerged fermentation was studied. In solid-state fermentation, polyurethane foam was used as an inert support, and corncob, corn leaves, and broken corn were used as biodegradable supports. The highest xylanase activity was produced in the medium with xylan in both fermentation systems, reaching 18,020 U/L and 19,266 U/L for submerged and solid-state fermentation, respectively. Cellulase production was observed only in the culture medium with carboxymethylcellulose, obtaining values of 7,872 U/L in submerged fermentation and 9,439 U/L in solid-state fermentation. The acid protease was produced only in minimal medium with glucose in acidic pH, reaching the highest levels of activity in SSF (806 U/L. The corncob was the best biodegradable support for the production of xylanases and acid protease. Two isoenzymes of xylanase and cellulase were observed in both fermentation systems, and three isoenzymes of xylanase were produced on the biodegradable supports.

  1. Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

    Science.gov (United States)

    Christoff, Ana Paula; Passaia, Gisele; Salvati, Caroline; Alves-Ferreira, Márcio; Margis-Pinheiro, Marcia; Margis, Rogerio

    2016-09-01

    Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

  2. Inactivation of cystein-aspartic acid protease (caspase)-1 by saikosaponin A.

    Science.gov (United States)

    Han, Na-Ra; Kim, Hyung-Min; Jeong, Hyun-Ja

    2011-01-01

    This work investigates the anti-inflammatory mechanism of saikosaponin A (SA), a major component of Bupleurum falcatum LINNE. SA significantly inhibited phorbol myristate acetate (PMA) plus A23187-induced the production and expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human mast cell (HMC)-1 cells. SA suppressed PMA plus A23187-induced phosphorylation of extracellular signal-regulated kinase and p38. When HMC-1 cells were treated with SA, translocation of nuclear factor (NF)-κB/Rel A into nucleus and degradation of inhibitor of NF-κB (IκB) in cytoplasm were inhibited. SA decreased PMA plus A23187-induced cysteine-aspartic acid protease (caspase)-1 activity. IL-1β production was also inhibited by SA. Finally, SA significantly decreased the number of nasal rubs and serum TNF-α level in the ovalbumin-sensitized allergic rhinitis mouse model. The underlying mechanism involves, at least in part, inactivation of caspase-1, which provides new evidence for therapeutic application of SA to target inflammatory processes.

  3. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

    Science.gov (United States)

    Almonte, Antoine G; Qadri, Laura H; Sultan, Faraz A; Watson, Jennifer A; Mount, Daniel J; Rumbaugh, Gavin; Sweatt, J David

    2013-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity. © 2012 International Society for Neurochemistry.

  4. Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids.

    Science.gov (United States)

    Dragovich, P S; Webber, S E; Prins, T J; Zhou, R; Marakovits, J T; Tikhe, J G; Fuhrman, S A; Patick, A K; Matthews, D A; Ford, C E; Brown, E L; Binford, S L; Meador, J W; Ferre, R A; Worland, S T

    1999-08-02

    Tripeptide-derived molecules incorporating N-methyl amino acid residues and C-terminal Michael acceptor moieties were evaluated as irreversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). Such compounds displayed good 3CP inhibition activity (k(obs)/[I] up to 610,000 M(-1) s(-1)) and potent in vitro antiviral properties (EC50 approaching 0.03 microM) when tested against HRV serotype-14.

  5. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles.

    Science.gov (United States)

    Elhenawy, Wael; Debelyy, Mykhaylo O; Feldman, Mario F

    2014-03-11

    Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion

  6. Modulating fatty acid oxidation in heart failure.

    Science.gov (United States)

    Lionetti, Vincenzo; Stanley, William C; Recchia, Fabio A

    2011-05-01

    In the advanced stages of heart failure, many key enzymes involved in myocardial energy substrate metabolism display various degrees of down-regulation. The net effect of the altered metabolic phenotype consists of reduced cardiac fatty oxidation, increased glycolysis and glucose oxidation, and rigidity of the metabolic response to changes in workload. Is this metabolic shift an adaptive mechanism that protects the heart or a maladaptive process that accelerates structural and functional derangement? The question remains open; however, the metabolic remodelling of the failing heart has induced a number of investigators to test the hypothesis that pharmacological modulation of myocardial substrate utilization might prove therapeutically advantageous. The present review addresses the effects of indirect and direct modulators of fatty acid (FA) oxidation, which are the best pharmacological agents available to date for 'metabolic therapy' of failing hearts. Evidence for the efficacy of therapeutic strategies based on modulators of FA metabolism is mixed, pointing to the possibility that the molecular/biochemical alterations induced by these pharmacological agents are more complex than originally thought. Much remains to be understood; however, the beneficial effects of molecules such as perhexiline and trimetazidine in small clinical trials indicate that this promising therapeutic strategy is worthy of further pursuit.

  7. Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

    Science.gov (United States)

    Watanabe, Masanori; Techapun, Charin; Kuntiya, Ampin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Takenaka, Shinji; Maeda, Isamu; Koyama, Masahiro; Nakamura, Kozo

    2017-02-01

    A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

  8. Cysteine proteases and acid phosphatases contribute to Tetrahymena spp. pathogenicity in guppies, Poecilia reticulata.

    Science.gov (United States)

    Leibowitz, M Pimenta; Ofir, R; Golan-Goldhirsh, A; Zilberg, D

    2009-12-03

    Systemic tetrahymenosis caused by the protozoan parasite Tetrahymena spp. is a serious problem in guppy (Poecilia reticulata) farms worldwide. There is no therapeutic solution for the systemic form of this disease. Guppies severely infected with Tetrahymena spp. were imported by a commercial ornamental fish farm and brought to our laboratory. Tetrahymena sp. (Tet-NI) was isolated and in vitro cultured. Isolates maintained in culture for different time periods (as reflected by different numbers of passages in culture) were analyzed-Tet-NI 1, 4, 5 and 6, with Tet-NI 1 being cultured for the longest period (about 15 months, 54 passages) and Tet-NI 6 for the shortest (2.5 months, 10 passages). Controlled internal infection was successfully achieved by IP injection with most isolates, except for Tet-NI 1 which produced no infection. The isolate Tet-NI 6 induced the highest infection rates in internal organs (80% vs. 50% and 64% for Tet-NI 4 and 5, respectively) and mortality rates (67% vs. 20% and 27% for Tet-NI 4 and 5, respectively, and 6.7% for Tet-NI 1). The correlation between pathogenicity and Tetrahymena enzymatic activity was studied. Electrophoretic analyses revealed at least two bands of gelanolytic activity in Tet-NI 4 and 5, three bands in Tet-NI 6, and no activity in Tet-NI 1. Total inhibition of gelanolytic activity was observed after pretreatment of Tet-NI 6 with E-64, a highly selective cysteine protease inhibitor. Using hemoglobin as a substrate, Tet-NI 6 had two bands of proteolytic activity and no bands were observed in Tet-NI 1. A correlation was observed between pathogenicity and acid phosphatase activities (analyzed by commercial fluorescence kit) for Tet-NI 1 and Tet-NI 6.

  9. 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.

  10. Production of Bioactive Peptides from Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease

    Directory of Open Access Journals (Sweden)

    Naifu Wang

    2014-10-01

    Full Text Available In this study, soybean meal was first solid state fermented with different strains of Lactic Acid Bacteria (LAB. Among the strains used, Lactobacillus plantarum Lp6 was selected for further studies because of its highest Degree of Hydrolysis (DH of protein (2.49±0.08% in soybean meal after 72 h fermentation. Soybean meal fermented with L. plantarum Lp6 can also improve its DPPH radical scavenging and Angiotensin Converting Enzyme (ACE inhibitory activities. The addition of protease into soybean meal during the fermentation resulted in lowered IC50 of DPPH radical scavenging and ACE inhibitory activities, indicating more bioactive peptides were produced during fermentation. Molecular weight distribution analysis revealed the Extracts from Fermented Soybean Meal (EFSM was mainly composed of oligopeptides. These results indicated that soybean meal fermented with L. plantarum Lp6 and protease could be an easy and cheap method to produce functional food.

  11. Brain-derived neurotrophic factor inducing angiogenesis through modulation of matrix-degrading proteases

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Recent studies have proved that brain-derived neurotrophic factor (BDNF) possesses angiogenic activity in vitro and in vivo. However, the proangiogenic mechanism of BDNF has not yet been provided with enough information. To explore the proangiogenic mechanism of BDNF, we investigated the effects of BDNF on extracellular proteolytic enzymes, including matrix metalloproteinases (MMPs) and serine proteases, particularly the urokinase-type plasminogen activator (uPA)-plasmin system in human umbilical vein endothelial cells (HUVECs) model. Methods Tube formation assay was performed in vitro to evaluate the effects of BDNF on angiogenesis. The HUVECs were treated with various concentrations of BDNF (25-400 ng/ml) for different (6-48 hours), reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA in HUVECs, and the conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively. uPA, plasminogen activator inhibitor (PAI)-1, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 were quantified by western blotting analysis. Results BDNF elicited robust and elongated angiogeneis in two-dimensional cultures of HUVECs in comparison with control. The stimulation of serum-starved HUVECs with BDNF caused obvious increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation. However, BDNF had no effect on TIMP-1 and TIMP-2 production. BDNF increased uPA and PAI-1 production in a dose-dependent manner. Maximal activation of uPA and PAI-1 expression in HUVECs was induced by 100 ng/ml BDNF, while effects of 200 ng/ml and 400 ng/ml BDNF were slightly reduced in comparison with with those of 100 ng/ml. Protease activity for uPA was also increased by BDNF in a dose-dependent manner. BDNF also stimulated uPA and PAI-1 production beyond that in control cultures in a time

  12. 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 potenti

  13. The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

    Science.gov (United States)

    Pantaléon, Véronique; Soavelomandroso, Anna Philibertine; Bouttier, Sylvie; Briandet, Romain; Roxas, Bryan; Chu, Michele; Collignon, Anne; Janoir, Claire; Vedantam, Gayatri; Candela, Thomas

    2015-01-01

    Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

  14. The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

    Directory of Open Access Journals (Sweden)

    Véronique Pantaléon

    Full Text Available Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

  15. Sentrin/SUMO specific proteases as novel tissue-selective modulators of vitamin D receptor-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Wai-Ping Lee

    Full Text Available Vitamin D receptor (VDR is a substrate for modification with small ubiquitin-like modifier (SUMO. To further assess the role of reversible SUMOylation within the vitamin D hormonal response, we evaluated the effects of sentrin/SUMO-specific proteases (SENPs that can function to remove small ubiquitin-like modifier (SUMO from target proteins upon the activities of VDR and related receptors. We report that SENP1 and SENP2 strikingly potentiate ligand-mediated transactivation of VDR and also its heterodimeric partner, retinoid X receptor (RXRα with depletion of cellular SENP1 significantly diminishing the hormonal responsiveness of the endogenous vitamin D target gene CYP24A1. We find that SENP-directed modulation of VDR activity is cell line-dependent, achieving potent modulatory effects in Caco-2 and HEK-293 cells, while in MCF-7 cells the vitamin D signal is unaffected by any tested SENP. In support of their function as novel modulators of the vitamin D hormonal pathway we demonstrate that both SENP1 and SENP2 can interact with VDR and reverse its modification with SUMO2. In a preliminary analysis we identify lysine 91, a residue known to be critical for formation and DNA binding of the VDR-RXR heterodimer, as a minor SUMO acceptor site within VDR. In combination, our results support a repressor function for SUMOylation of VDR and reveal SENPs as a novel class of VDR/RXR co-regulatory protein that significantly modulate the vitamin D response and which could also have important impact upon the functionality of both RXR-containing homo and heterodimers.

  16. Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

    Directory of Open Access Journals (Sweden)

    Laetitia Aerts

    Full Text Available Human metapneumovirus (hMPV infection causes acute respiratory tract infections (RTI which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1 and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

  17. The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host.

    Science.gov (United States)

    Bhullar, Kirandeep; Zarepour, Maryam; Yu, Hongbing; Yang, Hong; Croxen, Matthew; Stahl, Martin; Finlay, B Brett; Turvey, Stuart E; Vallance, Bruce A

    2015-07-01

    Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed "protein involved in colonization," or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (ΔpicC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ΔpicC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ΔpicC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicC phenotype. Exploring other aspects of PicC function, the ΔpicC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ΔpicC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ΔpicC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC's major role in vivo may be to limit C. rodentium's stimulation of the host's innate immune system.

  18. Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast.

    Science.gov (United States)

    Lei, Hongjie; Zheng, Liye; Wang, Chenxia; Zhao, Haifeng; Zhao, Mouming

    2013-02-01

    The objective of this study was to investigate the changes in free amino acids (FAA) composition by supplementing three commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of wort mashing, and monitoring the effects on the assimilation pattern of FAA and fermentation performance of lager yeast (Saccharomyces pastorianus) during normal and high gravity fermentations. Proteases supplementation significantly improved the extract yield and FAA level of mashed worts. Normal gravity worts treated with Flavorzyme and Neutrase exhibited higher fermentability, ethanol production and flavor volatiles concentration compared to the control worts, while these beneficial effects were observed in high gravity worts treated with Protamex and Neutrase. The reason for the above results is proposed to be the change in the assimilation pattern of FAA in lager yeast with increased wort gravity, especially for the improved assimilation ratios of Leu, Arg, Phe, His, Asp and Val. In normal gravity fermentations, there were strong correlations between the assimilation amounts of Lys, Leu, Arg and His and fermentability, while in high gravity fermentations, these good correlations were found with only Lys and His. The present study suggested that optimizing the composition of FAA by supplementing proteases during wort mashing was beneficial to beer brewing for improving fermentation performance of lager yeast and flavor volatiles formation.

  19. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

    Science.gov (United States)

    Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

    2016-03-01

    The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Modulation of dendritic cell function and immune response by cysteine protease inhibitor from murine nematode parasite Heligmosomoides polygyrus.

    Science.gov (United States)

    Sun, Yanxia; Liu, Guiyun; Li, Zhaotao; Chen, Yue; Liu, Yunfeng; Liu, Boyu; Su, Zhong

    2013-04-01

    Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively and the cysteine protease inhibitor (CPI or cystatin) is identified as one of the major immunomodulators. In the present study, we cloned and produced recombinant CPI protein from the murine nematode parasite Heligmosomoides polygyrus (rHp-CPI) and investigated its immunomodulatory effects on dendritic cell (DC) function and immune responses in mice. Bone-marrow-derived CD11c(+) DC (BMDC) that were exposed to rHp-CPI during the differentiation stage showed reduced MHC-II molecule expression compared with BMDC that were generated in normal culture conditions. The BMDC generated in the presence of rHp-CPI also exhibited reduced expression of CD40, CD86 and MHC-II molecules and reduced interleukin-6 and tumour necrosis factor-α cytokine production when stimulated with Toll-like receptor ligand CpG. Activation of BMDC generated in normal conditions induced by lipopolysaccharide and CpG was also suppressed by rHp-CPI, as shown by reduced co-stimulatory molecule expression and cytokine production. Furthermore, BMDC treated with rHp-CPI before ovalbumin (OVA) antigen pulsing induced a weaker proliferation response and less interferon-γ production of OVA-specific CD4(+) T cells compared with BMDC without rHp-CPI pre-treatment. Adoptive transfer of rHp-CPI-treated and OVA-loaded BMDC to mice induced significantly lower levels of antigen-specific antibody response than the BMDC loaded with antigen alone. These results demonstrated that the CPI from nematode parasites is able to modulate differentiation and activation stages of BMDC. It also interferes with antigen and MHC-II molecule processing and Toll-like receptor signalling pathway, resulting in functionally deficient DC that induce a suboptimum immune response.

  1. β-Amino acid catalyzed asymmetric Michael additions: design of organocatalysts with catalytic acid/base dyad inspired by serine proteases.

    Science.gov (United States)

    Yang, Hui; Wong, Ming Wah

    2011-09-16

    A new type of chiral β-amino acid catalyst has been computationally designed, mimicking the enzyme catalysis of serine proteases. Our catalyst approach is based on the bioinspired catalytic acid/base dyad, namely, a carboxyl and imidazole pair. DFT calculations predict that this designed organocatalyst catalyzes Michael additions of aldehydes to nitroalkenes with excellent enantioselectivities and remarkably high anti diastereoselectivities. The unusual stacked geometry of the enamine intermediate, hydrogen bonding network, and the adoption of an exo transition state are the keys to understand the stereoselectivity.

  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. 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...

  4. Screening of protease producing fungi for microbial digestion of seed proteins and synthesis of amino acids-metalnutrient chelates.

    Science.gov (United States)

    Deore, G B; Limaye, A S; Dushing, Y A; Dhobale, S B; Kale, S; Laware, S L

    2013-01-15

    The problem of metalnutrient deficiency is becoming more serious with the introduction of modern agricultural practices. As a result, metalnutrient deficiency is recognized as one of the critical yield limiting factors. Metalnutrients are generally offered in their sulphate or oxide forms. However, it is reported that organically bound minerals generally have a higher bioavailability than inorganic minerals. Chelation makes otherwise unavailable metalnutrients plant available. Amino acids are well known among various chelating agents. In present investigation the fungus Paecilomyces variotii PR-4 was isolated from soil and was used for production of protease and determination of its activity. Proteins from germinating seeds of chick pea, mung bean, soybean and cowpea were hydrolyzed for the production of amino acids. Amino acids were recovered, estimated and utilized for chelation of metalnutrients viz., Zn, Cu, Fe, Mn, Mg, B and Mo. The resultant chelates were employed to detect with Fourier Transform Infra-Red Spectrophotometer (FTIR) analysis. The peaks of most intensive bands in the IR spectra of ligands recorded were present in the intervals of the wave numbers 3500-3300 and 1720-1700 cm(-1). Chelation of metalnutrients led to the broadening of peak and changes of the peak position of hydroxyl groups, which indicated the binding of the carboxylic groups and primary amine groups of amino acids to the metalnutrients. The resultant amino acids-metalnutrient chelates can be utilized as organic fertilizer.

  5. Effects of dietary soybean stachyose and phytic acid on gene expressions of serine proteases in Japanese flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Mi, Haifeng; Mai, Kangsen; Zhang, Wenbing; Wu, Chenglong; Cai, Yinghua

    2011-09-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder ( Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively. Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY ( P0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  6. Fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus aspartic protease, an Aspergillus aspartic protease per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a protease of the aspartic proteinase-type,

  7. 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...

  8. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused...... to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N...... 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. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence.

    Science.gov (United States)

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

    Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (kcat/Km) in a wide range of temperatures, mainly due to an increase of k1 (substrate diffusion) and k2 (acylation) for V149I and of k2 and k3 (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔGa°) of activation for thermal denaturation may govern the thermostability. The value of ΔGa° is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.

  10. Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain.

    Science.gov (United States)

    Meissner, Lena; Kauffmann, Kira; Wengeler, Timo; Mitsunaga, Hitoshi; Fukusaki, Eiichiro; Büchs, Jochen

    2015-09-01

    Bacillus spp. are used for the production of industrial enzymes but are also known to be capable of producing biopolymers such as poly(γ-glutamic acid). Biopolymers increase the viscosity of the fermentation broth, thereby impairing mixing, gas/liquid mass and heat transfer in any bioreactor system. Undesired biopolymer formation has a significant impact on the fermentation and downstream processing performance. This study shows how undesirable poly(γ-glutamic acid) formation of an industrial protease producing Bacillus licheniformis strain was prevented by switching the nitrogen source from ammonium to nitrate. The viscosity was reduced from 32 to 2.5 mPa s. A constant or changing pH value did not influence the poly(γ-glutamic acid) production. Protease production was not affected: protease activities of 38 and 46 U mL(-1) were obtained for ammonium and nitrate, respectively. With the presented results, protease production with industrial Bacillus strains is now possible without the negative impact on fermentation and downstream processing by undesired poly(γ-glutamic acid) formation.

  11. 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...... 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....

  12. A plasma membrane association module in yeast amino acid transporters

    NARCIS (Netherlands)

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-01-01

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in sili

  13. A plasma membrane association module in yeast amino acid transporters

    NARCIS (Netherlands)

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-01-01

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in

  14. Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    MI Haifeng; MAI Kangsen; ZHANG Wenbing; WU Chenglong; CAI Yinghua

    2011-01-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish.The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus).These genes are trypsinogen 1 (poTRY),elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY).Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00,0.40,0.80 and 1.50%),4 levels of PA (0.00,0.20,0.40 and 0.80),respectively.Japanese flounder (initial weight 2.45 g±0.01 g)were fed with these diets for 10 weeks with three replications per treatment.At the end of 10 weeks,supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression ofpoTRY and poCTRY (P<0.05).The same level of dietary SBS significantly decreased the gene expression of poEL.In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%)significantly decreased the gene expression ofpoTRY,poCTRY and poEL (P<0.05).However,excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05).These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder,and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  15. A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity

    Science.gov (United States)

    Li, Bingqing; Yue, Yingying; Zhang, Yajie; Yuan, Zenglin; Li, Peng; Song, Nannan; Lin, Wei; Liu, Yan; Gu, Lichuan; Meng, Hong

    2017-01-01

    Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae. PMID:28217559

  16. 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.

  17. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

    The present invention relates to a polypeptide exhibiting a protease inhibitory activity and uses of said polypeptide in methods for inhibiting, directly or indirectly, one or more proteases of the blood clotting cascade. The invention also relates to use of said polypeptide as a pharmaceutical e...

  18. 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.

  19. Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

    Science.gov (United States)

    Nassal, M; Galle, P R; Schaller, H

    1989-01-01

    The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases. Images PMID:2657101

  20. Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

    Science.gov (United States)

    Hidayat, Habibi

    2017-03-01

    16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

  1. Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors.

    Science.gov (United States)

    Weigel, Lena F; Nitsche, Christoph; Graf, Dominik; Bartenschlager, Ralf; Klein, Christian D

    2015-10-01

    Dengue virus is an increasingly global pathogen. One of the promising targets for antiviral drug discovery against dengue and related flaviviruses such as West Nile virus is the viral serine protease NS2B-NS3. We here report the synthesis and in vitro characterization of potent peptidic inhibitors of dengue virus protease that incorporate phenylalanine and phenylglycine derivatives as arginine-mimicking groups with modulated basicity. The most promising compounds were (4-amidino)-L-phenylalanine-containing inhibitors, which reached nanomolar affinities against dengue virus protease. The type and position of the substituents on the phenylglycine and phenylalanine side chains has a significant effect on the inhibitory activity against dengue virus protease and selectivity against other proteases. In addition, the non-natural, basic amino acids described here may have relevance for the development of other peptidic and peptidomimetic drugs such as inhibitors of the blood clotting cascade.

  2. 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.

  3. Ubiquitin specific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells.

    Science.gov (United States)

    Nepal, Saroj; Shrestha, Anup; Park, Pil-Hoon

    2015-09-05

    Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, respectively in cancer cells. Ubiquitin specific protease-2 (USP-2), a deubiquitinating enzyme, is known to impair proteasome-induced degradation of cyclin D1, a critical cell cycle regulator. Herein, we investigated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expression both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyclin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together, our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regulation by adipokines. Thus, USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines.

  4. Modulating the electronic structure of amino acids: interaction of model lewis acids with anthranilic acid

    Directory of Open Access Journals (Sweden)

    Tareq Irshaidat

    2014-01-01

    Full Text Available On the basis of theoretical B3LYP calculations, Yáñez and co-workers (J. Chem. Theory Comput. 2012, 8, 2293 illustrated that beryllium ions are capable of significantly modulating (changing the electronic structures of imidazole. In this computational organic chemistry study, the interaction of this β-amino acid and five model Lewis acids (BeF1+, Be2+, AlF2(1+, AlF2+, and Al3+ were investigated. Several aspects were addressed: natural bond orbitals, including second order perturbation analysis of intra-molecular charge delocalization and the natural population analysis atomic charges; molecular geometries; selected infrared stretching frequencies (C-N, C-O, and N-H, and selected ¹H-NMR chemical shifts. The data illustrate that this interaction can weaken the H-O bond and goes beyond strengthening the intra-molecular hydrogen bond (N...H-O to cause a spontaneous transfer of the proton to the nitrogen atom in five cases generating zwitterion structures. Many new features are observed. Most importantly, the zwitterion structures include a stabilizing hydrogen bond (N-H...O that varies in relative strength according to the Lewis acid. These findings explain the experimental observations of α-amino acids (for example: J. Am. Chem. Soc. 2001, 123, 3577 and are the first reported fundamental electronic structure characterization of β-amino acids in zwitterion form.

  5. T Helper 1 and T Helper 2 Cytokines Differentially Modulate Expression of Filaggrin and its Processing Proteases in Human Keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Zheng-Hong Di; Lei Ma; Rui-Qun Qi; Xiao-Dong Sun; Wei Huo; Li Zhang; Ya-Ni Lyu

    2016-01-01

    Background: Atopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th 1/Th2) cytokine expression.Filaggrin (FLG) is the key protein to maintaining skin barrier function.Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes.However, the role ofThl/Th2 cytokines on FLG processing is not substantially documented.Our aim was to investigate the impact ofThl/Th2 cytokines on FLG processing.Methods: HaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ(IFN-γ).FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting.Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry.Results: IL-4/13 significantly reduced, while IFN-γsignificantly up-regulated FLG expression.IL-4/13 significantly increased, whereas IFN-γsignificantly decreased the expression ofkallikreins 5 and 7, matriptase and channel-activating serine protease 1.On the contrary, IL-4/13 significantly decreased, while IFN-γincreased the expression of LEKTI and caspase-14.Similar trends were observed in AD lesions.Conclusions: Our results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes.The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.

  6. Modulating gene function with peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Crooke, Stanley T.

    2008-01-01

    A review on peptide nucleic acid (PNA) oligomers as modulators of gene expression ranging from gene silencing at the mRNAor the dsDNA (antigene) level, and redirection of mRNA splicing to gene activation through transcription bubble mimicking. PNA chem., anti-infective agents, cellular delivery, ...

  7. Conformation of a coarse-grained protein chain (an aspartic acid protease) model in effective solvent by a bond-fluctuating Monte Carlo simulation

    Science.gov (United States)

    Pandey, R. B.; Farmer, B. L.

    2008-03-01

    In a coarse-grained description of a protein chain, all of the 20 amino acid residues can be broadly divided into three groups: Hydrophobic (H) , polar (P) , and electrostatic (E) . A protein can be described by nodes tethered in a chain with a node representing an amino acid group. Aspartic acid protease consists of 99 residues in a well-defined sequence of H , P , and E nodes tethered together by fluctuating bonds. The protein chain is placed on a cubic lattice where empty lattice sites constitute an effective solvent medium. The amino groups (nodes) interact with the solvent (S) sites with appropriate attractive (PS) and repulsive (HS) interactions with the solvent and execute their stochastic movement with the Metropolis algorithm. Variations of the root mean square displacements of the center of mass and that of its center node of the protease chain and its gyration radius with the time steps are examined for different solvent strength. The structure of the protease swells on increasing the solvent interaction strength which tends to enhance the relaxation time to reach the diffusive behavior of the chain. Equilibrium radius of gyration increases linearly on increasing the solvent strength: A slow rate of increase in weak solvent regime is followed by a faster swelling in stronger solvent. Variation of the gyration radius with the time steps suggests that the protein chain moves via contraction and expansion in a somewhat quasiperiodic pattern particularly in strong solvent.

  8. 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)

  9. Targets for modulation of fatty acid oxidation in the heart.

    Science.gov (United States)

    Lopaschuk, Gary D

    2004-03-01

    Fatty acids are a major source of fuel used by the heart to provide large amounts of energy necessary to sustain contractile function. In the healthy heart, a balance between fatty acid and carbohydrate use ensures that energy supply to the heart matches demand. However, myocardial ischemia causes profound changes in metabolism, including alterations in glucose and fatty acid metabolism that can lead to excessive myocardial fatty acid oxidation, which occurs at the expense of glucose oxidation. This contributes to cellular acidosis, a decrease in cardiac efficiency and contractile dysfunction in the ischemic heart. Inhibition of fatty acid oxidation has recently emerged as a promising approach to the prevention of these adverse effects of fatty acids. As a result, a number of key enzymes involved in the metabolism of fatty acids are potential targets for therapeutic intervention in myocardial ischemia. This includes inhibition of fatty acid uptake into the myocyte, inhibition of mitochondrial fatty acid uptake and direct inhibition of fatty acid beta-oxidation. This review describes these potential targets for modulation of energy metabolism in the heart.

  10. Fatty acids as modulators of neutrophil recruitment, function and survival.

    Science.gov (United States)

    Rodrigues, Hosana G; Takeo Sato, Fabio; Curi, Rui; Vinolo, Marco A R

    2016-08-15

    Neutrophils are well-known to act in the destruction of invading microorganisms. They have also been implicated in the activation of other immune cells including B- and T-lymphocytes and in the resolution of inflammation and tissue regeneration. Neutrophils are produced in the bone marrow and released into the circulation from where they migrate to tissues to perform their effector functions. Neutrophils are in constant contact with fatty acids that can modulate their function, activation and fate (survival or cell death) through different mechanisms. In this review, the effects of fatty acids pertaining to five classes, namely, long-chain saturated fatty acids (LCSFAs), short-chain fatty acids (SCFAs), and omega-3 (n-3), omega-6 (n-6) and omega-9 (n-9) unsaturated fatty acids, on neutrophils and the relevance of these effects for disease development are discussed.

  11. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    Science.gov (United States)

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  12. 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.

  13. 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

  14. Expression of acid protease from Aspergillus kawachii in Aspergillus niger%白曲霉酸性蛋白酶在黑曲霉中表达

    Institute of Scientific and Technical Information of China (English)

    李杰; 吴婷; 马南; 王欣; 杨建乐; 李健友; 张会

    2016-01-01

    研究分析固态发酵酸性蛋白酶生产菌株及其产品,ITS序列鉴定结果表明,该菌株为曲霉属,质谱分析结果表明其产品为Aspergillus saitoi酸性蛋白酶Aspergillopepsin I(EC.3.4.23.18)。根据Aspergillus saitoi酸性蛋白酶Aspergillopepsin I基因序列pep1(GI:473517)设计引物,以固态发酵酸性蛋白酶生产菌株基因组DNA为模板,利用PCR技术扩增获得pep1基因。序列分析结果表明,扩增片段与白曲霉Aspergillus kawachii酸性蛋白酶基因组序列相似性为99%,其编码蛋白与白曲霉Aspergillus kawachii酸性蛋白酶(GAA90749.1)相似性为100%,将该基因命名为pepB。构建黑曲霉表达载体pSZHG-pepB,通过农杆菌介导法转化黑曲霉CICC2462,筛选得到在glaA位点发生同源重组纯合转化子。经摇瓶发酵后,对产物进行SDS-PAGE、酶活检测以及酸性蛋白酶酶学性质和酶稳定性研究。结果表明,纯合同源重组菌株经SDS-PAGE检测时在47 ku左右处有明显目的蛋白条带,其发酵产物酸性蛋白酶酶活达5543 U·mL-1,为出发菌株152倍。对菌株所产酸性蛋白酶酶学性质研究发现,该酶最适反应温度为50℃,最适反应pH 3.0,在4℃和25℃条件下,酶在pH 3.0~4.0时较稳定。%In this study, solid fermentation acid protease's producing strain from Zhaodong Richeng enzyme Ltd. and its products were analyzed , ITS sequence indicated that the strain belongs to the genus Aspergillus and mass spectrometric analysis showed that its product was acidic protease Aspergillopepsin I (EC.3.4.23.18) of Aspergillus saitoi. According to gene sequence of pep1 (GI:473517) from acid protease Aspergillopepsin I of Aspergillus saitoi, primers were designed, producing strain's genomic DNA from Solid-state fermentation acid protease as the template, and by the method of PCR, pep1 gene was cloned. Sequence analysis showed that amplified fragment was 99%similar to acid protease

  15. 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.

  16. Probing the segmental mobility and energy of the active zones of a protein chain (aspartic acid protease) by a coarse-grained bond-fluctuation Monte Carlo simulation

    Science.gov (United States)

    Pandey, Ras; Farmer, Barry

    2008-03-01

    A protein chain such as aspartic acid protease is described by a specific sequence of 99 residues each with its own specific characteristics. In a coarse-grained description, the backbone of a protein chain is described by nodes tethered together by peptide bonds where each node (the amino acid group) is characterized by molecular weight and hydrophobicity. A well-developed and somewhat mature computational modeling tool for the polymer chain such as the bond-fluctuation model is used to study such a specific protein chain with its constitutive amino groups and their sequence. The relative magnitude of hydrophobicity is used to develop appropriate interaction potentials for these amino acid groups in explicit solvent. The Metropolis algorithm is used to move each node and solvent constituent. Local energy and mobility of each amino group are analyzed along with global energy, mobility, and conformation of the protein chain. Effect of the solvent interaction and its concentration on these quantities will be presented.

  17. Protective role of Kalpaamruthaa in type II diabetes mellitus-induced cardiovascular disease through the modulation of protease-activated receptor-1

    Directory of Open Access Journals (Sweden)

    Raja Latha

    2015-01-01

    Full Text Available Background: Kalpaamruthaa (KA is a formulatory herbal preparation has beneficial antioxidant, anti-apoptotic and anti-inflammatory properties against cardiovascular damage (CVD. Objective: The present study was undertaken to investigate the protective role of KA in type II diabetes mellitus-induced CVD through the modulation of protease-activated receptor-1 (PAR1. Materials and Methods: CVD was developed in 8 weeks after type II diabetes mellitus induction with high fat diet (2 weeks and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 h interval. CVD-induced rats treated with KA (200 mg/kg b.w. in 0.5 ml of olive oil orally for 4 weeks. Results: KA increased the activities of enzymatic antioxidants and the levels of non-enzymatic antioxidants in pancreas of CVD-induced rats. KA effectively reduced the lipid peroxides and carbonyl content in the pancreas of CVD-induced rats. KA reduced cellular damage by ameliorating the activities of marker enzymes in plasma, heart and liver. The protective nature of KA was further evidenced by histological observation in pancreas. Further, KA reduced CVD by decreasing the expression of PAR1 in heart. Conclusion: This study exhibits the defending role of KA in type II diabetes mellitus-induced CVD through altering PAR1.

  18. Glycolic acid modulates the mechanical property and degradation of poly(glycerol, sebacate, glycolic acid).

    Science.gov (United States)

    Sun, Zhi-Jie; Wu, Lan; Huang, Wei; Chen, Chang; Chen, Yan; Lu, Xi-Li; Zhang, Xiao-Lan; Yang, Bao-Feng; Dong, De-Li

    2010-01-01

    The development of biodegradable materials with controllable degradation properties is beneficial for a variety of applications. Poly(glycerol-sebacate) (PGS) is a promising candidate of biomaterials; so we synthesize a series of poly(glycerol, sebacate, glycolic acid) (PGSG) with 1:2:0, 1:2:0.2, 1:2:0.4, 1:2:0.6, 1:2:1 mole ratio of glycerol, sebacate, and glycolic acid to elucidate the relation of doped glycolic acid to the degradation rate and mechanical properties. The microstructures of the polymers with different doping of glycolic acid were dissimilar. PGSG with glycolic acid in the ratio of 0.2 displayed an integral degree of ordering, different to those with glycolic acid in the ratio of 0, 0.4, 0.6, and 1, which showed mild phase separation structure. The number, DeltaH(m), and temperature of the PGSG melting peaks tended to decrease with the increasing ratio of doped glycolic acid. In vitro and in vivo degradation tests showed that the degradation rate of PGSG with glycolic acid in the ratio of 0.2 was slowest, but in the ratio range of 0, 0.4, and 0.6, the degradation rate increased with the increase of glycolic acid. All PGSG samples displayed good tissue response and anticoagulant effects. Our data suggest that doping glycolic acid can modulate the microstructure and degree of crosslinking of PGS, thereby control the degradation rate of PGS.

  19. Prospective and randomised evaluation of the protease-modulating effect of oxidised regenerated cellulose/collagen matrix treatment in pressure sore ulcers

    NARCIS (Netherlands)

    Kloeters, O.; Unglaub, F.; Laat, E. de; Abeelen, M. Van; Ulrich, D.

    2016-01-01

    In chronic wounds, excess levels and activity of proteases such as elastase and plasmin have been detected. Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) has been reported to ameliorate the wound microenvironment by binding and inactivating excess proteases in wound exudates.

  20. Journey describing the discoveries of anti-HIV triterpene acid families targeting HIV-entry/fusion, protease functioning and maturation stages.

    Science.gov (United States)

    Patel, Rahul V; Park, Se Won

    2014-01-01

    HIV infection/AIDS, is a fatal disease multiplying rapidly in virtually every country. Extensive creations are in progress to arrest the replication of the HIV, following the destruction of either particular step involved in the progression of HIV infection. In such endeavors, mechanistically more diverse antiviral therapies were showcased using naturally occurring triterpene acid and their derivatives acting at various stages of HIV life cycle like entry or fusion, function of HIV protease enzyme and finally at maturation. The present article holds an extensive step-by-step summary of anti-HIV breakthroughs of triterpene acid analogues and their derivatives with synthetic and activity aspects, featuring fertile clues for novel anti-HIV drug design, which helps to develop unprecedented opportunities to discover the next-generation anti- HIV armamentarium.

  1. 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.

  2. 酸性蛋白酶在山西老陈醋酿造中的应用%Appl ication of Acid Protease in Brewing of Shanxi Mature Vinegar

    Institute of Scientific and Technical Information of China (English)

    潘佩平; 王慕华; 苏槟楠; 蔡颖慧; 赵玉明; 李海涛

    2014-01-01

    To determine the effect of acid protease activity of Daqu on the brewing of Shanxi mature vinegar,the brewing process of Shanxi mature vinegar is simulated in laboratory.Acid protease is added to improve Daqu enzyme activity.The results show that acid protease promotes the utilization of raw material during alcohol fermentation period and increases alcohol and amino acid nitrogen yield. The optimal acid protease activity is 501.5 U/g,while the alcohol production rate increases by 20.9%, reaching its highest value,and amino acid nitrogen content increases by 232.7%.The mature vinegar applied acid protease has higher total acid content with good clarity and stable quality.%为探明大曲中酸性蛋白酶酶活对山西老陈醋酿造的影响,在实验室内模拟山西老陈醋酿造工艺,以额外添加酸性蛋白酶来提高大曲酶活。结果发现:酸性蛋白酶可以在酒精发酵阶段促进原料的利用,提高酒精及氨基酸态氮含量,酒精度达到最高值的最适酸性蛋白酶酶活为501.5 U/g,此时,酒精产率提高20.9%,氨基酸态氮含量提高232.7%。通过醋酸发酵制得的老陈醋总酸含量高、澄清度好、品质稳定。

  3. Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC.

    Science.gov (United States)

    Wetzel, Daniela; Fischer, Ralf-Jörg

    2015-11-01

    Small acid-soluble proteins (SASPs) play an important role in protection of DNA in dormant bacterial endospores against damage by heat, UV radiation or enzymic degradation. In the genome of the strict anaerobe Clostridium acetobutylicum, five genes encoding SASPs have been annotated and here a further sixth candidate is suggested. The ssp genes are expressed in parallel dependent upon Spo0A, a master regulator of sporulation. Analysis of the transcription start points revealed a σG or a σF consensus promoter upstream of each ssp gene, confirming a forespore-specific gene expression. SASPs were termed SspA (Cac2365), SspB (Cac1522), SspD (Cac1620), SspF (Cac2372), SspH (Cac1663) and Tlp (Cac1487). Here it is shown that with the exception of Tlp, every purified recombinant SASP is able to bind DNA in vitro thereby protecting it against enzymic degradation by DNase I. Moreover, SspB and SspD were specifically cleaved by the two germination-specific proteases GPR (Cac1275) and YyaC (Cac2857), which were overexpressed in Escherichia coli and activated by an autocleavage reaction. Thus, for the first time to our knowledge, GPR-like activity and SASP specificity could be demonstrated for a YyaC-like protein. Collectively, the results assign SspA, SspB, SspD, SspF and SspH of C. acetobutylicum as members of α/β-type SASPs, whereas Tlp seems to be a non-DNA-binding spore protein of unknown function. In acetic acid-extracted proteins of dormant spores of C. acetobutylicum, SspA was identified almost exclusively, indicating its dominant biological role as a major α/β-type SASP in vivo.

  4. Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins.

    Science.gov (United States)

    Chen, Xin; Oshima, Tadayuki; Tomita, Toshihiko; Fukui, Hirokazu; Watari, Jiro; Matsumoto, Takayuki; Miwa, Hiroto

    2011-08-01

    Experimental models for esophageal epithelium in vitro either suffer from poor differentiation or complicated culture systems. An air-liquid interface system with normal human bronchial epithelial cells can serve as a model of esophageal-like squamous epithelial cell layers. Here, we explore the influence of bile acids on barrier function and tight junction (TJ) proteins. The cells were treated with taurocholic acid (TCA), glycocholic acid (GCA), or deoxycholic acid (DCA) at different pH values, or with pepsin. Barrier function was measured by transepithelial electrical resistance (TEER) and the diffusion of paracellular tracers (permeability). The expression of TJ proteins, including claudin-1 and claudin-4, was examined by Western blotting of 1% Nonidet P-40-soluble and -insoluble fractions. TCA and GCA dose-dependently decreased TEER and increased paracellular permeability at pH 3 after 1 h. TCA (4 mM) or GCA (4 mM) did not change TEER and permeability at pH 7.4 or pH 4. The combination of TCA and GCA at pH 3 significantly decreased TEER and increased permeability at lower concentrations (2 mM). Pepsin (4 mg/ml, pH 3) did not have any effect on barrier function. DCA significantly decreased the TEER and increased permeability at pH 6, a weakly acidic condition. TCA (4 mM) and GCA (4 mM) significantly decreased the insoluble fractions of claudin-1 and claudin-4 at pH 3. In conclusion, acidic bile salts disrupted the squamous epithelial barrier function partly by modulating the amounts of claudin-1 and claudin-4. These results provide new insights for understanding the role of TJ proteins in esophagitis.

  5. Gastric and intestinal proteases resistance of chicken acidic chitinase nominates chitin-containing organisms for alternative whole edible diets for poultry.

    Science.gov (United States)

    Tabata, Eri; Kashimura, Akinori; Wakita, Satoshi; Ohno, Misa; Sakaguchi, Masayoshi; Sugahara, Yasusato; Kino, Yoshihiro; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

    2017-07-27

    Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), functions as a major structural component in crustaceans, insects and fungi and is the second most abundant polysaccharide in the nature. Although these chitin-containing organisms have been suggested as novel animal feed resources, chitin has long been considered as indigestible fibers in the animal body. Recently, we reported that acidic chitinase (Chia) is a protease-resistant major glycosidase in mouse gastrointestinal tract (GIT) and that it digests chitin in the mouse stomach. However, the physiological role of Chia in other animals including poultry remains unknown. Here, we report that Chia can function as a digestive enzyme that breaks down chitin-containing organisms in chicken GIT. Chia mRNA is predominantly expressed in the glandular stomach tissue in normal chicken. We also show that chicken Chia has a robust chitinolytic activity at pH 2.0 and is highly resistant to proteolysis by pepsin and trypsin/chymotrypsin under conditions mimicking GIT. Chia degraded shells of mealworm larvae in the presence of digestive proteases and produced (GlcNAc)2. Thus, functional similarity of chicken Chia with the mouse enzyme suggests that chitin-containing organisms can be used for alternative poultry diets not only as whole edible resources but also as enhancers of their nutritional value.

  6. Scores of amino acid 0D-3D information as applied in cleavage site prediction and better specificity elucidation for human immunodeficiency virus type 1 protease

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A new set of descriptors,namely score vectors of the zero dimension,one dimension,two dimensions and three dimensions(SZOTT),was derived from principle component analysis of a matrix of 1369 structural variables including 0D,1D,2D and 3D information for the 20 coded amino acids. SZOTT scales were then used in cleavage site prediction of human immunodeficiency virus type 1 protease. Linear discriminant analysis(LDA) and support vector machines(SVM) were applied to developing models to predict the cleavage sites. The results obtained by linear discriminant analysis(LDA) and support vector machines(SVM) are as follows. The Matthews correlation coefficients(MCC) by the resubstitution test,leave-one-out cross validation(LOOCV) and external validation are 0.879 and 0.911,0.849 and 0.901,0.822 and 0.846,respectively. The receiver operating characteristic(ROC) analysis showed that the SVM model possesses better simulative and predictive ability in comparison with the LDA model. Satisfactory results show that SZOTT descriptors can be further used to predict cleavage sites of human immunodeficiency virus type 1 protease.

  7. Scores of amino acid 0D-3D information as applied in cleavage site prediction and better specificity elucidation for human immunodeficiency virus type 1 protease

    Institute of Scientific and Technical Information of China (English)

    KANG LiFang; LIANG GuiZhao; SHU Mao; YANG ShanBin; LI ZhiLiang

    2008-01-01

    A new set of descriptors, namely score vectors of the zero dimension, one dimension, two dimensions and three dimensions (SZOTT), was derived from principle component analysis of a matrix of 1369 structural variables including 0D, 1D, 2D and 3D information for the 20 coded amino acids. SZOTT scales were then used in cleavage site prediction of human immunodeficiency virus type 1 protease. Linear discriminant analysis (LDA) and support vector machines (SVM) were applied to developing models to predict the cleavage sites. The results obtained by linear discriminant analysis (LDA) and support vector machines (SVM) are as follows. The Matthews correlation coefficients (MCC) by the resubstitution test, leave-one-out cross validation (LOOCV) and external validation are 0.879 and 0.911, 0.649 and 0.901, 0.822 and 0.846, respectively. The receiver operating characteristic (ROC) analysis showed that the SVM model possesses better simulative and predictive ability in comparison with the LDA model. Satisfactory results show that SZOTT descriptors can be further used to predict cleavage sites of human immunodeficiency virus type 1 protease.

  8. Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD

    Science.gov (United States)

    Aly, Khaled A; Beebe, Emily T; Chan, Chi H; Goren, Michael A; Sepúlveda, Carolina; Makino, Shin-ichi; Fox, Brian G; Forest, Katrina T

    2013-01-01

    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence. PMID:23255525

  9. Effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage.

    Science.gov (United States)

    Yuan, XianJun; Wen, AiYou; Desta, Seare T; Dong, ZhiHao; Shao, Tao

    2017-07-01

    Short-chain fatty salts have been widely used as food and forage preservatives because of their antimicrobial properties. This study evaluated the effects of four chemical compounds with antimicrobial properties on nitrogen transformations and intrinsic protease activity of alfalfa silage. Potassium diformate (PD) and formic acid (FA) rapidly reduced silage pH. Silages treated with sodium diacetate (SD) and calcium propionate (CAP) had higher final peptide N concentrations than other silage. The free amino acid N contents in PD and FA treated silages were lower than other silages at all intervals of ensilage. The ammonia N concentrations in FA and PD silages were the lowest, followed by SD and CAP silages. As ensiling progressed, the aminopeptidase activity was completely lost by day 5 for FA and PD silages and inactive by day 7 for SD silage, while it remained active after day 7 for control and CAP silage. The carboxypeptidase activities in FA and PD silages were already reduced below 50% by day 1 of ensiling. Potassium diformate was as effective as formic acid in depressing the proteolysis, while sodium diacetate and calcium propionate were inferior to formic acid in protecting alfalfa proteins from being hydrolysed. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  10. Association between nasal carriage of Staphylococcus aureus and the human complement cascade activator serine protease C1 inhibitor (C1INH) valine vs. methionine polymorphism at amino acid position 480.

    NARCIS (Netherlands)

    Emonts, M.; Jongh, C.E. de; Houwing-Duistermaat, J.J.; Leeuwen, W.B. van; Groot, R. de; Verbrugh, H.A.; Hermans, P.W.M.; Belkum, A. van

    2007-01-01

    Staphylococcus aureus produces compounds that interfere with complement deposition. We hypothesized that humans have developed countermeasures to staphylococcal complement evasion and we screened for single nucleotide polymorphisms in the serine protease C1 inhibitor (C1INH) gene at amino acid posit

  11. Proteases as Insecticidal Agents

    OpenAIRE

    Robert L. Harrison; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic...

  12. Amino acid starvation induced by protease inhibition produces differential alterations in redox status and the thiol proteome in organogenesis-stage rat embryos and visceral yolk sacs.

    Science.gov (United States)

    Harris, Craig; Jilek, Joseph L; Sant, Karilyn E; Pohl, Jan; Reed, Matthew; Hansen, Jason M

    2015-12-01

    The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26-h period (GD10-GD11) and 50 μM over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual

  13. Poly(isophthalic acid)(ethylene oxide) as a Macromolecular Modulator for Metal-Organic Polyhedra.

    Science.gov (United States)

    Chen, Teng-Hao; Wang, Le; Trueblood, Jonathan V; Grassian, Vicki H; Cohen, Seth M

    2016-08-03

    A new strategy was developed by using a polymer ligand, poly(isophthalic acid)(ethylene oxide), to modulate the growth of metal-organic polyhedra (MOP) crystals. This macromolecular modulator can effectively control the crystal habit of several different Cu24L24 (L = isophthalic acid derivatives) MOPs. The polymer also directed the formation of MOP structures under reaction conditions that only produce metal-organic frameworks in the absence of modulator. Moreover, the polymer also enabled the deposition of MOP crystals on glass surfaces. This macromolecular modulator strategy provides an innovative approach to control the morphology and assembly of MOP particles.

  14. Synthesis and evaluation of non-peptidic cysteine protease inhibitors of P. falciparum derived from etacrynic acid.

    Science.gov (United States)

    Dude, Marie-Adrienne; Kaeppler, Ulrich; Herb, Monika; Schiller, Markus; Schulz, Franziska; Vedder, Birgit; Heppner, Saskia; Pradel, Gabriele; Gut, Jiri; Rosenthal, Philip J; Schirmeister, Tanja; Leippe, Matthias; Gelhaus, Christoph

    2008-12-23

    A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC(50) values of 9.0 and 18.8 microM against Plasmodia.

  15. Synthesis and Evaluation of Non-peptidic Cysteine Protease Inhibitors of P. falciparum Derived from Etacrynic Acid

    Directory of Open Access Journals (Sweden)

    Christoph Gelhaus

    2008-12-01

    Full Text Available A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC50 values of 9.0 and 18.8 μM against Plasmodia.

  16. Synthesis and Evaluation of Non-peptidic Cysteine Protease Inhibitors of P. falciparum Derived from Etacrynic Acid

    OpenAIRE

    Christoph Gelhaus; Matthias Leippe; Tanja Schirmeister; Rosenthal, Philip J.; Jiri Gut; Gabriele Pradel; Saskia Heppner; Birgit Vedder; Franziska Schulz,; Markus Schiller; Monika Herb; Ulrich Kaeppler; Marie-Adrienne Dude

    2008-01-01

    A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC50 values of 9.0 and 18.8 μM against Plasmodia.

  17. Acid protease and formation of multiple forms of glucoamylase in batch and continuous cultures of Aspergillus niger

    DEFF Research Database (Denmark)

    Aalbæk, Thomas; Reeslev, Morten; Jensen, Bo;

    2002-01-01

    In order to identify factors responsible for production of multiple forms of glucoamylase (GA) by Aspergillus niger Bo-1, the fungus was cultured in both complex and defined media in pH-controlled batch fermenters and chemostats. At all culture conditions three forms of GA were produced with mole......In order to identify factors responsible for production of multiple forms of glucoamylase (GA) by Aspergillus niger Bo-1, the fungus was cultured in both complex and defined media in pH-controlled batch fermenters and chemostats. At all culture conditions three forms of GA were produced...... with molecular weights of approx. 91 (GAI), 73 (GAII), and 59 kDa (GAIII). Data from batch fermentations with constant pH 3.0 and 5.0 showed a uniform distribution of extracellular GA forms throughout the fermentations and independent of culture growth phases. Furthermore, steady-state data from chemostat...... cultivations at constant pH 3.0 and 5.0 showed a similar distribution of extracellular GA forms and established that the nitrogen concentration of the medium (C/N ratio) did not affect the distribution of multiple forms of GA. The extracellular acid protease activity was only moderate when the fungus...

  18. Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

    1980-01-01

    ]oleate utilization were greater, relative to FABP concentrations, than in 60-d-old animals. The sex differences that characterize fatty acid utilization in adult rat hepatocytes are not present in cells from immature animals, and reflect in part the influence of sex steroids. It remains to be determined whether the observed relationship of hepatic FABP concentration to [14C]oleate utilization in adult cells is causal or secondary to changes in cellular fatty acid uptake effected through another mechanism. In either case, modulation of triglyceride-rich lipoprotein production by six steroids appears to be mediated to a significant extent by their effects on hepatic fatty acid utilization. PMID:7364935

  19. Modulation by Amino Acids: Toward Superior Control in the Synthesis of Zirconium Metal-Organic Frameworks.

    Science.gov (United States)

    Gutov, Oleksii V; Molina, Sonia; Escudero-Adán, Eduardo C; Shafir, Alexandr

    2016-09-12

    The synthesis of zirconium metal-organic frameworks (Zr MOFs) modulated by various amino acids, including l-proline, glycine, and l-phenylalanine, is shown to be a straightforward approach toward functional-group incorporation and particle-size control. High yields in Zr-MOF synthesis are achieved by employing 5 equivalents of the modulator at 120 °C. At lower temperatures, the method provides a series of Zr MOFs with increased particle size, including many suitable for single-crystal X-ray diffraction studies. Furthermore, amino acid modulators can be incorporated at defect sites in Zr MOFs with an amino acid/ligand ratio of up to 1:1, depending on the ligand structure and reaction conditions. The MOFs obtained through amino acid modulation exhibit an improved CO2 -capture capacity relative to nonfunctionalized materials. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Chlorogenic acid from honeysuckle improves hepatic lipid dysregulation and modulates hepatic fatty acid composition in rats with chronic endotoxin infusion.

    Science.gov (United States)

    Zhou, Yan; Ruan, Zheng; Wen, Yanmei; Yang, Yuhui; Mi, Shumei; Zhou, Lili; Wu, Xin; Ding, Sheng; Deng, Zeyuan; Wu, Guoyao; Yin, Yulong

    2016-03-01

    Chlorogenic acid as a natural hydroxycinnamic acid has protective effect for liver. Endotoxin induced metabolic disorder, such as lipid dysregulation and hyperlipidemia. In this study, we investigated the effect of chlorogenic acid in rats with chronic endotoxin infusion. The Sprague-Dawley rats with lipid metabolic disorder (LD group) were intraperitoneally injected endotoxin. And the rats of chlorogenic acid-LD group were daily received chlorogenic acid by intragastric administration. In chlorogenic acid-LD group, the area of visceral adipocyte was decreased and liver injury was ameliorated, as compared to LD group. In chlorogenic acid-LD group, serum triglycerides, free fatty acids, hepatic triglycerides and cholesterol were decreased, the proportion of C20:1, C24:1 and C18:3n-6, Δ9-18 and Δ6-desaturase activity index in the liver were decreased, and the proportion of C18:3n-3 acid was increased, compared to the LD group. Moreover, levels of phosphorylated AMP-activated protein kinase, carnitine palmitoyltransferase-I, and fatty acid β-oxidation were increased in chlorogenic acid-LD group compared to LD rats, whereas levels of fatty acid synthase and acetyl-CoA carboxylase were decreased. These findings demonstrate that chlorogenic acid effectively improves hepatic lipid dysregulation in rats by regulating fatty acid metabolism enzymes, stimulating AMP-activated protein kinase activation, and modulating levels of hepatic fatty acids.

  1. Inhibition of Clostridium botulinum 52A toxicity and protease activity by sodium acid pyrophosphate in media systems.

    OpenAIRE

    Wagner, M.K.; Busta, F. F.

    1985-01-01

    The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SA...

  2. Secreted fungal aspartic proteases: A review.

    Science.gov (United States)

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. Copyright © 2016 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  3. 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

    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.

  4. 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"...

  5. Modulating the selectivity of matriptase-2 inhibitors with unnatural amino acids.

    Science.gov (United States)

    St-Georges, Catherine; Désilets, Antoine; Béliveau, François; Ghinet, Mariana; Dion, Sébastien P; Colombo, Éloic; Boudreault, Pierre-Luc; Najmanovich, Rafael J; Leduc, Richard; Marsault, Éric

    2017-03-31

    Matriptase-2, a type II transmembrane serine protease (TTSP), is expressed in the liver and regulates iron homeostasis via the cleavage of hemojuvelin. Matriptase-2 emerges as an attractive target for the treatment of conditions associated with iron overload, such as hemochromatosis or beta-thalassemia. Starting from the crystal structure of its closest homolog matriptase, we constructed a homology model of matriptase-2 in order to further optimize the selectivity of serine trap peptidomimetic inhibitors for matriptase-2 vs matriptase. Careful modifications of the P4, P3 and P2 positions with the help of unnatural amino acids led to a thorough understanding of Structure-Activity Relationship and a >60-fold increase in selectivity for matriptase-2 vs matriptase. Additionally, the introduction of unnatural amino acids led to significant increases in plasma stability. Such compounds represent useful pharmacological tools to test matriptase-2 inhibition in a context of iron overload.

  6. Dietary Fatty Acids and Temperature Modulate Mitochondrial Function and Longevity in Drosophila

    Science.gov (United States)

    Rand, David M.

    2015-01-01

    Fluctuations in temperature and resource availability are conditions many organisms contend with in nature. Specific dietary nutrients such as fatty acids play an essential role in reproduction, cold adaptation, and metabolism in a variety of organisms. The present study characterizes how temperature and diet interact to modulate Drosophila physiology and life span. Flies were raised on media containing specific saturated, monounsaturated, or polyunsaturated fatty acids supplements at low concentrations and were placed in varied thermal environments. We found that dietary long-chain polyunsaturated fatty acids improve chill coma recovery and modulate mitochondrial function. Additionally, monounsaturated and polyunsaturated fatty acid food supplements were detrimental to life span regardless of temperature, and antioxidants were able to partially rescue this effect. This study provides insight into environmental modulation of Drosophila physiology and life span. PMID:25910846

  7. 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.

  8. 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.

  9. Prospective and randomised evaluation of the protease-modulating effect of oxidised regenerated cellulose/collagen matrix treatment in pressure sore ulcers.

    Science.gov (United States)

    Kloeters, Oliver; Unglaub, Frank; de Laat, Erik; van Abeelen, Marjolijn; Ulrich, Dietmar

    2016-12-01

    In chronic wounds, excess levels and activity of proteases such as elastase and plasmin have been detected. Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) has been reported to ameliorate the wound microenvironment by binding and inactivating excess proteases in wound exudates. In this study, the levels and activity of elastase and plasmin in wound exudates of pressure sore ulcers were measured to determine the beneficial effect of ORC/collagen matrix treatment compared with control treatment with a foam dressing. A total of 33 patients with pressure sores were enrolled in the study and were followed up for 12 weeks after treatment. Ten control patients were treated with a foam hydropolymer dressing (TIELLE(®) , Systagenix), and the remaining 23 patients were treated with ORC/collagen matrix plus the foam dressing (TIELLE(®) , Systagenix) on top. Wound assessments were carried out over 12 weeks on a weekly basis, with dressing changes twice a week. Ulcers were photographed and wound exudates were collected on admission and at days 5, 14 and then every 14 days to provide a visual record of any changes in appearance of the ulcer and healing rate and for biochemical analysis of the wound. The levels and activity of elastase and plasmin were measured in wound exudates. Statistical analysis was performed using ANOVA and Bonferroni's post hoc test with P-values pressure sore wounds showed a significant faster healing rate, which positively correlated with a decreased activity of elastase and plasmin in wound exudates. No signs of infection or intolerance to the ORC/collagen matrix were observed. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  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. Novel fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus serine protease of the subtilisin-type, an Aspergillus serine protease of the subtilisin-type per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a

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

    OpenAIRE

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

    2014-01-01

    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 ...

  13. 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.

  14. Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

    Science.gov (United States)

    Portovedo, Mariana; Ignacio-Souza, Letícia M; Bombassaro, Bruna; Coope, Andressa; Reginato, Andressa; Razolli, Daniela S; Torsoni, Márcio A; Torsoni, Adriana S; Leal, Raquel F; Velloso, Licio A; Milanski, Marciane

    2015-01-01

    Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

  15. Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

    Science.gov (United States)

    2012-10-14

    crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor. Biochemistry 42, 6709-6718...deacylation in cleavage of physiological sequences by the processing protease Kex2. Biochemistry 40, 3657-3665. (26) Rockwell, N. C., and Fuller, R. S

  16. Polyunsaturated fatty acids in the modulation of T-cell signalling.

    Science.gov (United States)

    Akhtar Khan, Naim

    2010-01-01

    n-3 polyunsaturated fatty acids (PUFA) have been shown to modulate immune responses. These agents, being considered as adjuvant immunosuppressants, have been used in the treatment of various inflammatory and autoimmune diseases. However, the molecular mechanisms of action of n-3 PUFA-induced immunosuppressive effects are not well-understood. Since exogenous n-3 PUFA, under in vitro and in vivo conditions, are efficiently incorporated into T-cell plasma membranes, a number of recent studies have demonstrated that these agents may modulate T-cell signalling. In this review, the interactions of n-3 PUFA with the second messenger cascade initiated during early and late events of T-cell activation are discussed. We particularly focus on how these fatty acids can modulate the production of diacylglycerol and the activation of protein kinase C, mitogen activated protein kinase, calcium signalling and translocation of transcriptional factors, implicated in the regulation of gene transcription in T-cells.

  17. 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.

  18. Modulation of polyepoxysuccinic acid on crystallization of calcium oxalate

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanqing [School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 210009 (China); Tang, Yongming, E-mail: tangym@njtech.edu.cn [School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 210009 (China); Xu, Jinqiu; Zhang, Dongqin [School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 210009 (China); Lu, Gang [College of Materials Science and Engineering, Nanjing Tech University, Nanjing 210009 (China); Jing, Wenheng [College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing 210009 (China); State Key Laboratory of Material-Oriented Chemical Engineering, Nanjing 210009 (China)

    2015-11-15

    The influence of polyepoxysuccinic acid (PESA) on the phase composition and crystal morphology of calcium oxalate was investigated in this paper. It was found that the presence of PESA inhibited the growth of the monoclinic calcium oxalate monohydrate (COM) crystal and promoted the nucleation of the tetragonal calcium oxalate dihydrate (COD). In addition, with the increase in PESA concentration, the aggregation of COD crystals was reduced but the particle size was increased. Under the conditions of low calcium-to-oxalate ratio and high CaOx concentration, PESA could not effectively stabilize the formation of COD. Based on molecular dynamic simulations, the adsorption of PESA on CaOx crystal faces was confirmed. - Graphical abstract: Introduction of PESA into crystallization solutions promotes the formation of calcium oxalate dehydrate and modifies the morphology of crystals. - Highlights: • PESA induces the formation of COD at low supersaturation. • Establishment of Ca-rich surface augments the adsorption of PESA. • At Ca/Ox=0.5 PESA cannot induce the formation of COD compared with Ca/Ox=2. • Interaction of PESA with COM faces is stronger than that with COD faces.

  19. Complex modulation of androgen responsive gene expression by methoxyacetic acid

    Directory of Open Access Journals (Sweden)

    Stanley Kerri A

    2011-03-01

    Full Text Available Abstract Background Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA, the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known. Methods A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale. Results MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes. Conclusions These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model.

  20. 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.

  1. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    Science.gov (United States)

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  2. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

    Science.gov (United States)

    Johnston, David B; McAloon, Andrew J

    2014-02-01

    The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

  3. Modulation of the Absorption Maximum of Rhodopsin by Amino Acids in the C-terminus†

    Science.gov (United States)

    Yokoyama, Shozo; Tada, Takashi; Yamato, Takahisa

    2008-01-01

    Vision begins when light is absorbed by visual pigments. It is commonly believed that the absorption spectra of visual pigments are modulated by interactions between the retinal and amino acids within or near 4.5 Å of the retinal in the transmembrane (TM) segments. However, this dogma has not been rigorously tested. In this study, we show that the retinal-opsin interactions extend well beyond the retinal binding pocket. We found that, although it is positioned outside of TM segments, the C-terminus of the rhodopsin in the rockfish longspine thornyhead (Sebastolobus altivelis) modulates its λmax by interacting mainly with the last TM segment. Our results illustrate how amino acids in the C-terminus are likely to interact with the retinal. We anticipate our analyses to be a starting point for viewing the spectral tuning of visual pigments as interactions between the retinal and key amino acids that are distributed throughout the entire pigment. PMID:16922606

  4. 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

    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...

  5. Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4.

    Science.gov (United States)

    Poulsen, M V; Vandenberg, R J

    2001-07-01

    1. The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were examined using radiolabelled substrate flux measurements and two-electrode voltage clamp techniques. 2. Niflumic acid significantly enhanced the substrate-gated currents in EAAT4, without affecting the affinity of the substrates towards EAAT4. At a concentration of 300 microM, niflumic acid caused a 19 +/- 5 % reduction in L-[(3)H]glutamate uptake and no significant effect on the uptake of DL-[(3)H]aspartate. Thus, enhancement of the substrate-gated currents in EAAT4 does not correlate with the rate of substrate transport and suggests that the niflumic acid-induced currents are not thermodynamically coupled to the transport of substrate. 3. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-expressing oocytes had similar functional consequences. However, niflumic acid still enhanced the L-glutamate-gated current to the same extent in the presence and absence of a saturating dose of arachidonic acid, which suggests that the sites of action of the two compounds are distinct. 4. The EAAT4-mediated currents for the two substrates, L-glutamate and L-aspartate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was not the same for the two substrates. Protons carry the L-glutamate-gated niflumic acid-induced current and both protons and chloride ions carry the L-aspartate-gated niflumic acid-induced current. 5. These results show that niflumic acid can be used to probe the functional aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflammatory drugs could be used as the basis for the development of novel modulators of glutamate transporters.

  6. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing....... These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  7. Modulation of arachidonic and linoleic acid metabolites in myeloperoxidase deficient mice during acute inflammation

    Science.gov (United States)

    Kubala, Lukas; Schmelzer, Kara R.; Klinke, Anna; Kolarova, Hana; Baldus, Stephan; Hammock, Bruce D.; Eiserich, Jason P.

    2010-01-01

    Acute inflammation is a common feature of many life-threatening pathologies, including septic shock. One hallmark of acute inflammation is the peroxidation of polyunsaturated fatty acids forming bioactive products, which regulate inflammation. Myeloperoxidase (MPO) is an abundant phagocyte-derived hemoprotein released during phagocyte activation. Here, we investigated the role of MPO in modulating biologically active arachidonic acid (AA) and linoleic acid (LA) metabolites during acute inflammation. Wild-type and MPO-knockout (KO) mice were exposed to intraperitoneally injected endotoxin for 24 h, and plasma LA and AA oxidation products were comprehensively analyzed using a liquid chromatography-mass spectrometry method. Compared to wild-type mice, MPO-KO mice had significantly lower plasma levels of LA epoxides and corresponding LA- and AA-derived fatty acid diols. AA and LA hydroxy intermediates (hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids) were also significantly lower in MPO-KO mice. Conversely, MPO-deficient mice had significantly higher plasma levels of cysteinyl-leukotrienes with well-known pro-inflammatory properties. In vitro experiments revealed significantly lower amounts of AA and LA epoxides, LA- and AA-derived fatty acid diols, and AA and LA hydroxy intermediates in stimulated polymorphonuclear neutrophils isolated from MPO-KO mice. Our results demonstrate that MPO modulates the balance of pro- and anti-inflammatory lipid mediators during acute inflammation. In this way, may control acute inflammatory diseases. PMID:20156554

  8. Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

    Science.gov (United States)

    Bernat, Viachaslau; Admas, Tizita Haimanot; Brox, Regine; Heinemann, Frank W; Tschammer, Nuska

    2014-11-21

    The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

  9. Hydrolysis of Fish Protein by Analkaline Protease

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

  10. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    António Rego

    2014-08-01

    Full Text Available Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria.

  11. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    Science.gov (United States)

    Rego, António; Duarte, Ana M.; Azevedo, Flávio; Sousa, Maria J.; Côrte-Real, Manuela; Chaves, Susana R.

    2014-01-01

    Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria. PMID:28357256

  12. Local atomic structure modulations activate metal oxide as electrocatalyst for hydrogen evolution in acidic water.

    Science.gov (United States)

    Li, Yu Hang; Liu, Peng Fei; Pan, Lin Feng; Wang, Hai Feng; Yang, Zhen Zhong; Zheng, Li Rong; Hu, P; Zhao, Hui Jun; Gu, Lin; Yang, Hua Gui

    2015-08-19

    Modifications of local structure at atomic level could precisely and effectively tune the capacity of materials, enabling enhancement in the catalytic activity. Here we modulate the local atomic structure of a classical but inert transition metal oxide, tungsten trioxide, to be an efficient electrocatalyst for hydrogen evolution in acidic water, which has shown promise as an alternative to platinum. Structural analyses and theoretical calculations together indicate that the origin of the enhanced activity could be attributed to the tailored electronic structure by means of the local atomic structure modulations. We anticipate that suitable structure modulations might be applied on other transition metal oxides to meet the optimal thermodynamic and kinetic requirements, which may pave the way to unlock the potential of other promising candidates as cost-effective electrocatalysts for hydrogen evolution in industry.

  13. 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.

  14. Peroxidase activity of bacterial cytochrome P450 enzymes: modulation by fatty acids and organic solvents.

    Science.gov (United States)

    Rabe, Kersten S; Erkelenz, Michael; Kiko, Kathrin; Niemeyer, Christof M

    2010-08-01

    The modulation of peroxidase activity by fatty acid additives and organic cosolvents was determined and compared for four bacterial cytochrome P450 enzymes, thermostable P450 CYP119A1, the P450 domain of CYP102A1 (BMP), CYP152A1 (P450(bsbeta)), and CYP101A1 (P450(cam)). Utilizing a high-throughput microplate assay, we were able to readily screen more than 100 combinations of enzymes, additives and cosolvents in a convenient and highly reproducible assay format. We found that, in general, CYP119A1 and BMP showed an increase in peroxidative activity in the presence of fatty acids, whereas CYP152A1 revealed a decrease in activity and CYP101A1 was only slightly affected. In particular, we observed that the conversion of the fluorogenic peroxidase substrate Amplex Red by CYP119A1 and BMP was increased by a factor of 38 or 11, respectively, when isopropanol and lauric acid were present in the reaction mixture. The activity of CYP119A1 could thus be modulated to reach more than 90% of the activity of CYP152A1 without effectors, which is the system with the highest peroxidative activity. For all P450s investigated we found distinctive reactivity patterns, which suggest similarities in the binding site of CYP119A1 and BMP in contrast with the other two proteins studied. Therefore, this study points towards a role of fatty acids as activators for CYP enzymes in addition to being mere substrates. In general, our detailed description of fatty acid- and organic solvent-effects is of practical interest because it illustrates that optimization of modulators and cosolvents can lead to significantly increased yields in biocatalysis.

  15. 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.

  16. Organochloride pesticides modulated gut microbiota and influenced bile acid metabolism in mice.

    Science.gov (United States)

    Liu, Qian; Shao, Wentao; Zhang, Chunlan; Xu, Cheng; Wang, Qihan; Liu, Hui; Sun, Haidong; Jiang, Zhaoyan; Gu, Aihua

    2017-07-01

    Organochlorine pesticides (OCPs) can persistently accumulate in body and threaten human health. Bile acids and intestinal microbial metabolism have emerged as important signaling molecules in the host. However, knowledge on which intestinal microbiota and bile acids are modified by OCPs remains unclear. In this study, adult male C57BL/6 mice were exposed to p, p'-dichlorodiphenyldichloroethylene (p, p'-DDE) and β-hexachlorocyclohexane (β-HCH) for 8 weeks. The relative abundance and composition of various bacterial species were analyzed by 16S rRNA gene sequencing. Bile acid composition was analyzed by metabolomic analysis using UPLC-MS. The expression of genes involved in hepatic and enteric bile acids metabolism was measured by real-time PCR. Expression of genes in bile acids synthesis and transportation were measured in HepG2 cells incubated with p, p'-DDE and β-HCH. Our findings showed OCPs changed relative abundance and composition of intestinal microbiota, especially in enhanced Lactobacillus with bile salt hydrolase (BSH) activity. OCPs affected bile acid composition, enhanced hydrophobicity, decreased expression of genes on bile acid reabsorption in the terminal ileum and compensatory increased expression of genes on synthesis of bile acids in the liver. We demonstrated that chronic exposure of OCPs could impair intestinal microbiota; as a result, hepatic and enteric bile acid profiles and metabolism were influenced. The findings in this study draw our attention to the hazards of chronic OCPs exposure in modulating bile acid metabolism that might cause metabolic disorders and their potential to cause related diseases in human. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Polyunsaturated Fatty Acids Differentially Modulate Cell Proliferation and Endocannabinoid System in Two Human Cancer Lines.

    Science.gov (United States)

    Gastón, Repossi; María Eugenia, Pasqualini; Das, Undurti N; Eynard, Aldo R

    2017-01-01

    Evidence suggests that quantity and quality of dietary polyunsaturated fatty acids (PUFAs) play a role in the development of cancer. However, the mechanisms involved in this interaction(s) are not clear. Endocannabinoids are lipid metabolites known to have growth modulatory actions. We studied the effect of supplementation with PUFAs ω-6 and ω-3 (essential fatty acids, EFAs), saturated and monounsaturated fatty acids (non-EFAs) on the growth of tumor cells and modifications in their endocannabinoid content. Cell cultures of human glioblastoma (T98G) and breast cancer (MCF7) were supplemented with 50 or 100 mmol EFAs and non-EFAs for 72 h. Cell proliferation was then determined by MTT, anandamide (AEA) levels by HPLC, total fatty acids profiles by GLC, CB1 receptor expression by WB and FAAH activity by spectrophotometric method. Fatty acids profile reflected the incorporation of the lipids supplemented in each assay. Arachidonic acid (EFA ω-6) supplementation increased AEA levels and inhibited the growth of T98G, whereas palmitic acid (non-EFA) enhanced their proliferation. In breast cancer (MCF7) cells, eicosapentaenoic acid (EFA ω-3) reduced and oleic acid (non-EFA) enhanced their proliferation. CB1 expression was higher in T98G and no differences were observed in FAAH activity. The growth of tumor cells can be differentially modulated by fatty acids and, at least in part, can be attributed to their ability to act on the components of the endocannabinoid system. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  18. Identification of a senescence-related protease in coriander leaves

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Senescence-related protease may play an important role in leaf senescence. By improved SDS-Gela- tin-PAGE assay, a 63 ku senescence-related protease (63 SRP) in coriander leaves was identified. Activity of 63 SRP was increased in parallel to the advance of coriander leaf senescence, and inhibited by treating the leaf with gibberellic acid, and enhanced by ethylene treatment. The 63 SRP was suggested to be a serine protease based on the fact that its activity was inhibited by the protease inhibitor PMSF. The optimal temperature for the activity of the 70 ku protease was 50℃. The maximal activity was observed at pH 6-9, some activity could be observed on the gel slices incubated at pH 5 or 11. The 63 SRP was partly purified by the way of ammonium sulfate precipitation and then gel slicing after gel electrophoresis.

  19. The maize cystatin CC9 interacts with apoplastic cysteine proteases.

    Science.gov (United States)

    van der Linde, Karina; Mueller, André N; Hemetsberger, Christoph; Kashani, Farnusch; van der Hoorn, Renier A L; Doehlemann, Gunther

    2012-11-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

  20. Divergent modulation of swine ileal microbiota by formic acid and methionine hydroxy analogue-free acid.

    Science.gov (United States)

    Apajalahti, J; Rademacher, M; Htoo, J K; Redshaw, M; Kettunen, A

    2009-06-01

    Management of intestinal microbiota of monogastric animals has increased in importance since the ban of growth promoting antibiotics in many countries. Organic acids have been used as alternatives to antibiotics by many feed manufacturers. Regardless of the wide usage, the effect, dose response and mode of action of acids on intestinal microbes is poorly understood. In this study, we investigated the effects of dietary supplementation of three commonly used products, namely formic acid (FA) (90%), dl-methionine (DLM) (99%) and liquid methionine hydroxy analogue-free acid (88%), on ileal microbiota of pigs. Laboratory simulation system, mimicking swine ileum, was used to study the products at various concentrations and combinations. Furthermore, selected combinations were tested in a piglet trial to confirm the findings made in in vitro studies. FA turned out to have a dual effect on ileal microbiota. At concentrations below 0.5%, it significantly stimulated bacteria, but at higher inclusion rates it was highly inhibitory. This finding, which was consistent in in vitro and in vivo studies, implies that reducing the dose of FA does not lead to a diluted inhibitory effect, but in fact, an opposite, stimulatory effect on intestinal microbiota. It is highly important that feed compounders acknowledge this finding. Unlike FA, the inhibitory effect of methionine hydroxy analogue on ileal bacteria was linearly dose dependent and significant at inclusion levels above 0.2%, in vitro. Partial replacement of methionine hydroxy analogue by FA, or FA by methionine hydroxy analogue, led to an unpredictable outcome due to the dual effects of FA; e.g., a minor inclusion of added FA changed the inhibitory effect of methionine hydroxy analogue into microbial stimulation by FA. Inhibition of ileal microbiota by methionine hydroxy analogue was detected only in in vitro studies, suggesting that intact methionine hydroxy analogue may not have reached the ileum, in live animals. Therefore

  1. Crystal structure of Spot 14, a modulator of fatty acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Colbert, Christopher L.; Kim, Chai-Wan; Moon, Young-Ah; Henry, Lisa; Palnitkar, Maya; McKean, William B.; Fitzgerald, Kevin; Deisenhofer, Johann; Horton, Jay D.; Kwon, Hyock Joo (Alnylam Pharm.); (UTSMC)

    2011-09-06

    Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 {angstrom} and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.

  2. 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.

  3. Fatty acid modulation of autoinducer (AI-2) influenced growth and macrophage invasion by Salmonella Typhimurium.

    Science.gov (United States)

    Widmer, Kenneth W; Jesudhasan, Palmy; Pillai, Suresh D

    2012-03-01

    Autoinducer-2 (AI-2) is a small molecule that is involved in bacterial cell-to-cell signaling whose precursor formation is mediated by luxS. A luxS mutant of Salmonella Typhimurium PJ002 (ΔluxS) was grown in glucose-containing M-9 minimal medium supplemented with varying concentrations (1×, 10×, and 100×) of long-chain fatty acids (linoleic acid, oleic acid, palmitic acid, and stearic acid) to study the influence of fatty acids on growth rate and macrophage invasion. Additionally, in vitro synthesized AI-2 was added to this medium to identify the influence of AI-2 on S. Typhimurium PJ002 (ΔluxS) growth rate and macrophage invasion. The growth rate constant (k) for each experimental treatment was determined based on OD₆₀₀ values recorded during 12 h of incubation. There was a significant (p=0.01) increase in the growth rate of S. Typhimurium PJ002 (ΔluxS) in the presence of AI-2 when compared to the phosphate-buffered saline (PBS) control. However, fatty acids either singly or in a mixture were unable to influence AI-2's effect on growth rate. The presence of AI-2 significantly (p=0.02) decreased the invasiveness of S. Typhimurium PJ002 (ΔluxS) towards the murine macrophage cell line, RAW 264.7. However, the fatty acid mixture was able to reverse this reduction and restore invasiveness to background levels. These results suggest that, while AI-2 may enhance the growth rate and reduce macrophage invasion by the luxS mutant S. Typhimurium PJ002 (ΔluxS), fatty acids may influence the virulence in S. Typhimurium (PJ002) by modulating AI-2 activity.

  4. Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca(2+)-release channels.

    Science.gov (United States)

    Oba, T

    1997-11-01

    The effects of niflumic acid on ryanodine receptors (RyRs) of frog skeletal muscle were studied by incorporating sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog muscle had two distinct types of RyRs in the SR: one showed a bell-shaped channel activation curve against cytoplasmic Ca2+ or niflumic acid, and its mean open probability (Po) was increased by perchlorate at 20-30 mM (termed "alpha-like" RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed "beta-like" RyR). The unitary conductance and reversal potential of both channel types were unaffected after exposure to niflumic acid when clamped at 0 mV. When clamped at more positive potentials, the beta-like RyR channel rectified this, increasing the unitary current. Treatment with niflumic acid did not inhibit the response of both channels to Ca2+ release channel modulators such as caffeine, ryanodine, and ruthenium red. The different effects of niflumic acid on Po and the unitary current amplitude in both types of channels may be attributable to the lack or the presence of inactivation sites and/or distinct responses to agonists.

  5. Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease fromArenicola cristata

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunling; JU Jiyu

    2015-01-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplifi-cation of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encod-ing a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (<40%) to other serine proteases. The gene encoding the active form ofA. cristata serine protease was cloned and expressed inE. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result sug-gested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clotin vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene inA. cristata.

  6. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    Science.gov (United States)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  7. Eicosapentaenoic acid modulates fatty acid metabolism and inflammation in Psammomys obesus.

    Science.gov (United States)

    Atek-Mebarki, Feriel; Hichami, Aziz; Abdoul-Azize, Souleymane; Bitam, Arezki; Koceïr, Elhadj Ahmed; Khan, Naim Akhtar

    2015-02-01

    The desert gerbil, Psammomys obesus, is a unique polygenic animal model of metabolic syndrome (insulin resistance, obesity and type 2 diabetes), and these pathological conditions resemble to those in human beings. In this study, the animals were fed ad libitum either a natural diet (ND) which contained desertic halophile plants or a standard laboratory diet (STD) or a diet which contained eicosapentaenoic acid (EPA), hence, termed as EPA diet (EPAD). In EPAD, 50% of total lipid content was replaced by EPA oil. By employing real-time PCR, we assessed liver expression of key genes involved in fatty acid metabolism such as PPAR-α, SREBP-1c, LXR-α and CHREBP. We also studied the expression of two inflammatory genes, i.e., TNF-α and IL-1β, in liver and adipose tissue of these animals. The STD, considered to be a high caloric diet for this animal, triggered insulin resistance and high lipid levels, along with high hepatic SREBP-1c, LXR-α and CHREBP mRNA expression. TNF-α and IL-1β mRNA were also high in liver of STD fed animals. Feeding EPAD improved plasma glucose, insulin and triacylglycerol levels along with hepatic lipid composition. These observations suggest that EPA exerts beneficial effects in P. obesus.

  8. Modulation of human stratum corneum properties by salicylic acid and all-trans-retinoic acid.

    Science.gov (United States)

    Piérard-Franchimont, C; Goffin, V; Piérard, G E

    1998-01-01

    Topical all-trans-retinoic acid (RA) has been reported to decrease the in vivo skin response to sodium lauryl sulfate (SLS). The converse was also shown with a synergistic effect of RA following prior applications of SLS. The reason for such effects is not clear. We employed measures of transepidermal water loss (TEWL), squamometry and sequential corneosurfametry to explore the protective activity of a 0.05% RA cream at the level of the stratum corneum. Nonionic oil-in-water emulsions with or without 5% salicylic acid (SA) served as test product references. Data indicated that the RA formulation was responsible for a stochastic impairment in the TEWL and for an increased intercorneocyte cohesion. SA and the unmedicated emulsion did not lead to similar TEWL changes. The squamometry test proved to be very sensitive to disclose the effects of SA and RA without, however, allowing to distinguish the difference in the physiological processes involved. The corneosurfametry bioassay did not show any protection or synergistic effect between RA or SA and SLS challenge on the stratum corneum. This is in contrast to a previous work showing a positive protective effect afforded by retinol against SLS. The combined effects of irritant compounds affecting the stratum corneum are complex. The precise reason for some of their biological consequences remains a conundrum. On balance, products such as SA and RA do not appear to afford protection or impairment to a surfactant challenge at the level of the stratum corneum.

  9. Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum.

    Science.gov (United States)

    Meevootisom, V; Niederpruem, D J

    1979-06-01

    The production of proteases was investigated during growth of dermatophytic fungi with special emphasis on Trichophyton rubrum. Exogenous glucose suppressed elastase production in all dermatophytes examined. The production of protease active guinea pig hair in keratin-salts broth by Microsporum gypseum. Trichophyton mentagrophytes and T. rubrum was also suppressed by glucose. Various carbohydrates added to keratin-salts broth curtailed protease production by T. rubrum as did individual amino acids but ammonium phosphate did not. Enzyme activities against guinea pig hair were compared in twenty-one diverse clinical isolates of T. rubrum cultured in keratin-salts broth. Activity also occurred towards casein, bovine serum albumin, keratin, collagen and elastin after keratin-growth. Studies concerning the properties of enzyme activities in culture filtrates of T. rubrum after keratin-growth suggested that multiple proteases occurred here. Hydrolysis of guinea pig hair and elastin were optimal at pH7 while keratinase was most active at alkaline pH. Divalent cations stimulated protease(s). Ferric ion and mercuric ion stimulated keratinase but were inhibitory to guinea pig hair hydrolysis and elastase. Chelating agents inhibited elastase and the hydrolysis of guinea pig hair more severely than keratinase and all of those effects were reversed by excess calcium. A serine-protease inhibitor, phenylmethylsulfonylfluoride (PMSF), curtailed keratinase but was less inhibitory to elastase and guinea pig hair hydrolysis. Soybean trypsin inhibitor arrested each protease.

  10. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate:Evidence of a protease horizontal gene transfer event

    Institute of Scientific and Technical Information of China (English)

    Cecilia Martinez-Rosales; Juan Jos Marizcurrena; Andrs Iriarte; Natalia Fullana; Hctor Musto; Susana Castro-Sowinski

    2015-01-01

    We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98%query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

  11. Proteases in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Ana Rita Sokolonski ANTON

    2006-09-01

    Full Text Available Introduction: The caries and the periodontal disease (PD are the most frequent alterations in the oral cavity. The PD presents two stages: gengivitis and periodontitis. The destruction of collagenous fibers which encases the tooth onto the alveolar bone is characteristic of the pariodontitis. The inclusion loss caused by this pathology is due to the presence of bacteria and their products, besides the tissue destruction. This process is caused by excessive discharge of cells of the organism defence which reach the damaged area, and among these cells are neutrophils. These cells free lysosomal granule, where enzymes known as proteases (elastase, colagenasis and catepsin G are present. When excessively delivered, they cause extensive tissue destruction. The organism innate defence respond to this process activating anti-proteases, such as alfa-1-antitripsin e alfa-2-macrogoblulin, and, as consequence, the inflammatory process is subdued. Objective: Revision of the literature on periodontitis and its markers. In periodontitis, the balance between protease and anti-protese seems to be altered and lead to the appearance of these ones. There is an increase of prevalence of PD in the world population. In recent times, it has been associated to systemic conditions that lead to tissue destruction. Perhaps, the cause is based on an exacerbated tissue reaction, more than on the bacterial aggression. Conclusion: The predisposition of the organism is an important factor for the disease development. At reading different studies, it was observed that the discharged protease during the neutrophils degranulation process has internal, not bacterial, origin.

  12. Death proteases come alive

    NARCIS (Netherlands)

    Woltering, E.J.

    2004-01-01

    Cell death in plants exhibits morphological features comparable to caspase-mediated apoptosis in animals, suggesting that plant cell death is executed by (caspase-like) proteases. However, to date, no caspase homologues have been identified in plants and therefore the existence and nature of these p

  13. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  14. 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.

  15. SAR studies on carboxylic acid series M(1) selective positive allosteric modulators (PAMs).

    Science.gov (United States)

    Kuduk, Scott D; Beshore, Douglas C

    2014-01-01

    There is mounting evidence from preclinical and early proof-of-concept studies suggesting that selective modulation of the M1 muscarinic receptor is efficacious in cognitive models of Alzheimer's disease (AD). A number of nonselective M1 muscarinic agonists have previously shown positive effects on cognitive function in AD patients, but were limited due to cholinergic adverse events thought to be mediated by pan activation of the M2 to M5 sub-types. Thus, there is a need to identify selective activators of the M1 receptor to evaluate their potential in cognitive disorders. One strategy to confer selectivity for M1 is the identification of allosteric agonists or positive allosteric modulators, which would target an allosteric site on the M1 receptor rather than the highly conserved orthosteric acetylcholine binding site. BQCA has been identified as a highly selective carboxylic acid M1 PAM and this review focuses on an extensive lead optimization campaign undertaken on this compound.

  16. Repurposing Resveratrol and Fluconazole To Modulate Human Cytochrome P450-Mediated Arachidonic Acid Metabolism.

    Science.gov (United States)

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2016-04-04

    Cytochrome P450 (P450) enzymes metabolize arachidonic acid (AA) to several biologically active epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Repurposing clinically-approved drugs could provide safe and readily available means to control EETs and HETEs levels in humans. Our aim was to determine how to significantly and selectively modulate P450-AA metabolism in humans by clinically-approved drugs. Liquid chromatography-mass spectrometry was used to determine the formation of 15 AA metabolites by human recombinant P450 enzymes, as well as human liver and kidney microsomes. CYP2C19 showed the highest EET-forming activity, while CYP1B1 and CYP2C8 showed the highest midchain HETE-forming activities. CYP1A1 and CYP4 showed the highest subterminal- and 20-HETE-forming activity, respectively. Resveratrol and fluconazole produced the most selective and significant modulation of hepatic P450-AA metabolism, comparable to investigational agents. Monte Carlo simulations showed that 90% of human population would experience a decrease by 6-22%, 16-39%, and 16-35% in 16-, 18-, and 20-HETE formation, respectively, after 2.5 g daily of resveratrol, and by 22-31% and 14-23% in 8,9- and 14,15-EET formation after 50 mg of fluconazole. In conclusion, clinically-approved drugs can provide selective and effective means to modulate P450-AA metabolism, comparable to investigational drugs. Resveratrol and fluconazole are good candidates to be repurposed as new P450-based treatments.

  17. 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

  18. Accurate prediction of HIV-1 drug response from the reverse transcriptase and protease amino acid sequences using sparse models created by convex optimization.

    Science.gov (United States)

    Rabinowitz, Matthew; Myers, Lance; Banjevic, Milena; Chan, Albert; Sweetkind-Singer, Joshua; Haberer, Jessica; McCann, Kelly; Wolkowicz, Roland

    2006-03-01

    Genotype-phenotype modeling problems are often overcomplete, or ill-posed, since the number of potential predictors-genes, proteins, mutations and their interactions-is large relative to the number of measured outcomes. Such datasets can still be used to train sparse parameter models that generalize accurately, by exerting a principle similar to Occam's Razor: When many possible theories can explain the observations, the most simple is most likely to be correct. We apply this philosophy to modeling the drug response of Type-1 Human Immunodeficiency Virus (HIV-1). Owing to the decreasing expense of genetic sequencing relative to in vitro phenotype testing, a statistical model that reliably predicts viral drug response from genetic data is an important tool in the selection of antiretroviral therapy (ART). The optimization techniques described will have application to many genotype-phenotype modeling problems for the purpose of enhancing clinical decisions. We describe two regression techniques for predicting viral phenotype in response to ART from genetic sequence data. Both techniques employ convex optimization for the continuous subset selection of a sparse set of model parameters. The first technique, the least absolute shrinkage and selection operator, uses the l(1) norm loss function to create a sparse linear model; the second, the support vector machine with radial basis kernel functions, uses the epsilon-insensitive loss function to create a sparse non-linear model. The techniques are applied to predict the response of the HIV-1 virus to 10 reverse transcriptase inhibitor and 7 protease inhibitor drugs. The genetic data are derived from the HIV coding sequences for the reverse transcriptase and protease enzymes. When tested by cross-validation with actual laboratory measurements, these models predict drug response phenotype more accurately than models previously discussed in the literature, and other canonical techniques described here. Key features of the

  19. Modulating effect of ascorbic Acid on transport-induced immunosuppression in goats.

    Science.gov (United States)

    Minka, Ndazo Salka; Ayo, Joseph Olusegun

    2011-01-01

    The effect of 12 h road transportation on some basic blood cells and the modulating role of ascorbic acid were investigated in 40 adult Red Sokoto goats during the hot dry season. The animals were divided into two groups, GI (experimental; n = 20) and GII (control; n = 20). Group 1 was administered with ascorbic acid (AA) per os at a dosage rate of 100 mg/kg body weight, while GII was given 10 mL of sterile water per goat. Forty minutes after the administration and loading, the goats were transported for 12 h. The result obtained in GII goats showed that loading, transportation, high ambient temperature (AT), and relative humidity (RH) encountered during transportation induced lymphopenia, neutrophilia, and eosinopenia, which can cause immunosuppression. In GI goats, the administration of AA prior to loading and transportation ameliorated the adverse effects of loading and transportation stress on neutrophil/lymphocyte ratio and eosinopenia of the goats.

  20. PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.

    Science.gov (United States)

    Lebeis, Sarah L; Paredes, Sur Herrera; Lundberg, Derek S; Breakfield, Natalie; Gehring, Jase; McDonald, Meredith; Malfatti, Stephanie; Glavina del Rio, Tijana; Jones, Corbin D; Tringe, Susannah G; Dangl, Jeffery L

    2015-08-21

    Immune systems distinguish "self" from "nonself" to maintain homeostasis and must differentially gate access to allow colonization by potentially beneficial, nonpathogenic microbes. Plant roots grow within extremely diverse soil microbial communities but assemble a taxonomically limited root-associated microbiome. We grew isogenic Arabidopsis thaliana mutants with altered immune systems in a wild soil and also in recolonization experiments with a synthetic bacterial community. We established that biosynthesis of, and signaling dependent on, the foliar defense phytohormone salicylic acid is required to assemble a normal root microbiome. Salicylic acid modulates colonization of the root by specific bacterial families. Thus, plant immune signaling drives selection from the available microbial communities to sculpt the root microbiome.

  1. The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons

    Directory of Open Access Journals (Sweden)

    Silverman Ann-Judith

    2002-02-01

    Full Text Available Abstract Background Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. Results Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1, and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. Conclusions These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

  2. Periodontal disease: modulation of the inflammatory cascade by dietary n-3 polyunsaturated fatty acids.

    Science.gov (United States)

    Sculley, D V

    2014-06-01

    Periodontal disease, including gingivitis and periodontitis, is caused by the interaction between pathogenic bacteria and the host immune system. The ensuing oxidative stress and inflammatory cascade result in the destruction of gingival tissue, alveolar bone and periodontal ligament. This article reviews the underlying mechanisms and host-bacteria interactions responsible for periodontal disease and evidence that nutritional supplementation with fish oil may provide a protective effect. Historical investigations of diet and disease have highlighted an inverse relationship between ingestion of fish oil, which is high in n-3 polyunsaturated fatty acids, and the incidence of typical inflammatory diseases such as arthritis and coronary heart disease. Ingestion of n-3 polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid, results in their incorporation into membrane phospholipids, which can alter eicosanoid production after stimulation during the immune response. These eicosanoids promote a reduction in chronic inflammation, which has led to the proposal that fish oil is a possible modulator of inflammation and may reduce the severity of periodontal diseases. Tentative animal and human studies have provided an indication of this effect. Further human investigation is needed to establish the protective effects of fish oil in relation to periodontal disease.

  3. Tempe consumption modulates fecal secondary bile acids, mucins, immunoglobulin A, enzyme activities, and cecal microflora and organic acids in rats.

    Science.gov (United States)

    Utama, Zaki; Okazaki, Yukako; Tomotake, Hiroyuki; Kato, Norihisa

    2013-06-01

    The present study investigated the effect of dietary tempe, a fermented soy product, on the colonic environment of rats fed high-fat (HF, 30 % fat; experiment 1) or low-fat (LF, 6 % fat; experiment 2) diets. Growing male rats were fed the experimental diets with or without 25 % tempe for 21 days. Tempe consumption slightly but significantly increased the growth of rats fed both the HF and LF diets (P fecal secondary bile acid, lithocholic acid (a risk factor of colon cancer) (P fecal mucins (indices of intestinal barrier function) and immunoglobulin A (IgA, an index of intestinal immune function) (P Analysis of the profile of cecal microflora revealed lower Bacteroides and higher Clostridium cluster XIVa levels in the tempe group of rats fed the HF diet (P fecal activity of β-glucosidase was markedly higher in the tempe group (P < 0.05), while that of urease was lower (P < 0.05) with both the HF and LF diets. The present results suggest that tempe consumption modulates the colonic environment in rats.

  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.

  5. Evolution of cyclic peptide protease inhibitors.

    Science.gov (United States)

    Young, Travis S; Young, Douglas D; Ahmad, Insha; Louis, John M; Benkovic, Stephen J; Schultz, Peter G

    2011-07-05

    We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X=pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ε-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

  6. Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating.

    Science.gov (United States)

    Yau, Hau-Jie; Baranauskas, Gytis; Martina, Marco

    2010-10-15

    The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

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

    Science.gov (United States)

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

    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.

  8. From proteases to proteomics.

    Science.gov (United States)

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  9. 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.

  10. Modulation of Ionotropic Glutamate Receptors and Acid-Sensing Ion Channels by Nitric Oxide

    Directory of Open Access Journals (Sweden)

    John Q Wang

    2012-05-01

    Full Text Available Ionotropic glutamate receptors (iGluR are ligand-gated ion channels and are densely expressed in broad areas of mammalian brains. Like iGluRs, acid-sensing ion channels (ASIC are ligand (H+-gated channels and are enriched in brain cells and peripheral sensory neurons. Both ion channels are enriched at excitatory synaptic sites, functionally coupled to each other, and subject to the modulation by a variety of signaling molecules. Central among them is a gasotransmitter, nitric oxide (NO. Available data show that NO activity-dependently modulates iGluRs and ASICs via either a direct or an indirect pathway. The former involves a NO-based and cGMP-independent posttranslational modification (S-nitrosylation of extracellular cysteine residues in channel subunits or channel-interacting proteins. The latter is achieved by NO activation of soluble guanylyl cyclase, which in turn triggers an intracellular cGMP-sensitive cascade to indirectly modulate iGluRs and ASICs. The NO modification is usually dynamic and reversible. Modified channels undergo significant, interrelated changes in biochemistry and electrophysiology. Since NO synthesis is enhanced in various neurological disorders, the NO modulation of iGluRs and ASICs is believed to be directly linked to the pathogenesis of these disorders. This review summarizes the direct and indirect modifications of iGluRs and ASICs by NO and analyzes the role of the NO-iGluR and NO-ASIC coupling in cell signaling and in the pathogenesis of certain related neurological diseases.

  11. Niflumic acid, a TRPV1 channel modulator, ameliorates stavudine-induced neuropathic pain.

    Science.gov (United States)

    Marwaha, Lovish; Bansal, Yashika; Singh, Raghunath; Saroj, Priyanka; Sodhi, Rupinder Kaur; Kuhad, Anurag

    2016-12-01

    TRP channels have been discovered as a specialized group of somatosensory neurons involved in the detection of noxious stimuli. Desensitization of TRPV1 located on dorsal root and trigeminal ganglia exhibits analgesic effect and makes it potential therapeutic target for treatment of neuropathic pain. With this background, the present study was aimed to investigate the protective effect of niflumic acid, a TRPV1 modulator, on stavudine (STV)-induced neuropathic pain in rats. Stavudine (50 mg/kg) was administered intravenously via tail vein in rats to induce neuropathic pain. Various behavioral tests were performed to access neuropathic pain (hyperalgesia and allodynia) on 7th, 14th, 21st, and 28th days. Electrophysiology (motor nerve conduction velocity; MNCV) and biochemical estimations were conducted after 28th day. Niflumic acid (10, 15, and 20 mg/kg) was administered intraperitoneally and evaluated against behavioral, electrophysiological (MNCV), and biochemical alterations in stavudine-treated rats. Pregabalin (30 mg/kg) was taken as reference standard and administered intraperitoneally. Four weeks after stavudine injection, rats developed behavioral, electrophysiological (MNCV), and biochemical (oxidative, nitrosative stress, and inflammatory cytokines, TRPV1) alterations. Niflumic acid restored core and associated symptoms of peripheral neuropathy by suppressing oxidative-nitrosative stress, inflammatory cytokines (TNF-α, IL-1β) and TRPV1 level in stavudine-induced neuropathic pain in rats. Pharmacological efficacy of niflumic acid (20 mg/kg) was equivalent to pregabalin (30 mg/kg). In conclusion, niflumic acid attenuates STV-induced behavioral, electrophysiological and biochemical alterations by manipulating TRP channel activity in two manners: (1) direct antagonistic action against TRPV1 channels and (2) indirect inhibition of TRP channels by blocking oxidative and inflammatory surge. Therefore, NA can be developed as a potential pharmacotherapeutic

  12. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    Science.gov (United States)

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  13. Generation and dietary modulation of anti-inflammatory electrophilic omega-3 fatty acid derivatives.

    Directory of Open Access Journals (Sweden)

    Chiara Cipollina

    Full Text Available Dietary ω-3 polyunsaturated fatty acids (PUFAs decrease cardiovascular risk via suppression of inflammation. The generation of electrophilic α,β-unsaturated ketone derivatives of the ω-3 PUFAs docosahexaenoic acid (DHA and docosapentaenoic acid (DPA in activated human macrophages is catalyzed by cyclooxygenase-2 (Cox-2. These derivatives are potent pleiotropic anti-inflammatory signaling mediators that act via mechanisms including the activation of Nrf2-dependent phase 2 gene expression and suppression of pro-inflammatory NF-κB-driven gene expression. Herein, the endogenous generation of ω-3 PUFAs electrophilic ketone derivatives and their hydroxy precursors was evaluated in human neutrophils. In addition, their dietary modulation was assessed through a randomized clinical trial.Endogenous generation of electrophilic omega-3 PUFAs and their hydroxy precursors was evaluated by mass spectrometry in neutrophils isolated from healthy subjects, both at baseline and upon stimulation with calcium ionophore. For the clinical trial, participants were healthy adults 30-55 years of age with a reported EPA+DHA consumption of ≤300 mg/day randomly assigned to parallel groups receiving daily oil capsule supplements for a period of 4 months containing either 1.4 g of EPA+DHA (active condition, n = 24 or identical appearing soybean oil (control condition, n = 21. Participants and laboratory technicians remained blinded to treatment assignments.5-lypoxygenase-dependent endogenous generation of 7-oxo-DHA, 7-oxo-DPA and 5-oxo-EPA and their hydroxy precursors is reported in human neutrophils stimulated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA. Dietary EPA+DHA supplementation significantly increased the formation of 7-oxo-DHA and 5-oxo-EPA, with no significant modulation of arachidonic acid (AA metabolite levels.The endogenous detection of these electrophilic ω-3 fatty acid ketone derivatives supports the precept that the

  14. Modulating membrane composition alters free fatty acid tolerance in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Rebecca M Lennen

    Full Text Available Microbial synthesis of free fatty acids (FFA is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10 that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.

  15. Ketogenic essential amino acids modulate lipid synthetic pathways and prevent hepatic steatosis in mice.

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    Yasushi Noguchi

    Full Text Available BACKGROUND: Although dietary ketogenic essential amino acid (KAA content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We designed a diet with a high ratio (E/N of essential amino acids (EAAs to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogenesis (DNL fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides. CONCLUSION: Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways.

  16. Cathepsin proteases in Toxoplasma gondii

    OpenAIRE

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and n...

  17. PROTEASES AND PROTEASE INHIBITORS INTERACTION: DEFENCE STRATEGY AGAINST

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE 1 N.J.CHIKHALE 2

    2014-12-01

    dissociates in short span of time (Oliva et. al., 2010. The activity of PI is to control proteolysis in cells and its components responsible for biochemical and physiological process by blocking, altering or preventing access to the enzyme active site. According to Broadway and Duffey (1986, the reduction in growth rate of insects is due to the hyperproduction of proteinases to compensate for loss of activity, which in turn led to the depletion of essential amino acids. Like other competitive PIs, cystatins form a light complex with target proteases to cause inhibition and interfere with dietary protein digestion in herbivores (Arai et. al., 2002. Chye et. al., (2006; Macedo et. al., (2004 and Ramos et. al., (2008 have demonstrated action of inhibitor proteins on insect gut proteases using artificial diets and invitro inhibition assays. KEY WORDS: Black kite, Trachea, Gross Anatomy.

  18. A tobacco etch virus protease with increased substrate tolerance at the P1' position.

    Science.gov (United States)

    Renicke, Christian; Spadaccini, Roberta; Taxis, Christof

    2013-01-01

    Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.

  19. Protease-mediated drug delivery

    Science.gov (United States)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  20. 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.

  1. Arachidonic acid modulates hippocampal calcium current via protein kinase C and oxygen radicals.

    Science.gov (United States)

    Keyser, D O; Alger, B E

    1990-10-01

    Arachidonic acid (AA) is a second messenger liberated via receptor activation of phospholipase A2 or diacylglycerol-lipase. We used whole-cell voltage clamp of acutely isolated hippocampal CA1 pyramidal cells to investigate the hypothesis that AA modulates Ca2+ channel current (ICa) via activation of protein kinase C (PKC) and generation of free radicals. AA depressed ICa in a dose- and time-dependent manner similar to that previously reported for the action of phorbol esters on ICa. A similar depression was seen with a xanthine-based free radical generating system. The specific PKC inhibitor PKCI (19-36), the protein kinase inhibitor H-7, and the superoxide free radical scavenger SOD each blocked ICa depression by 70%-80%. Complete block of the AA response occurred when SOD was used simultaneously with a PKC inhibitor. These data suggest that PKC and free radicals play a role in AA-induced suppression of ICa.

  2. Spatial self-phase modulation in the H2TPP(OH)4 doped in Boric Acid Glass

    CERN Document Server

    Allam, Srinivasa Rao; Venkatramaiah, N; Venkatesan, R; Sharan, Alok

    2015-01-01

    Self-diffraction rings or spatial self-phase modulation (SSPM) was observed in tetra-phenyl porphyrin derivative 5,10,15,20 - meso-tetrakis (4-hydroxyphenyl) porphyrin (H2TPP(OH)4) doped in boric acid glass (BAG) at 671 nm excitation wave-length lying within the absorption band of sample with TEM00 mode profile. Intensity modulated Z-scan was performed on these systems to study the thermal diffusion and to estimate the thermo-optic coefficients. The results obtained from self-diffraction rings experiment and modulated Z-scan are compared and analyzed for different concentration.

  3. 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.

  4. Boswellic acids target the human immune system-modulating antimicrobial peptide LL-37.

    Science.gov (United States)

    Henkel, Arne; Tausch, Lars; Pillong, Max; Jauch, Johann; Karas, Michael; Schneider, Gisbert; Werz, Oliver

    2015-12-01

    The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-β-BA (Aβ-BA) and 3-O-acetyl-11-keto-β-BA (AKβ-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 μM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders.

  5. ADAM Proteases and Gastrointestinal Function.

    Science.gov (United States)

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.

  6. ADAM Proteases and Gastrointestinal Function

    Science.gov (United States)

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  7. MicroRNA 146 (Mir146) modulates spermatogonial differentiation by retinoic acid in mice.

    Science.gov (United States)

    Huszar, Jessica M; Payne, Christopher J

    2013-01-01

    Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3' untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.

  8. MicroRNA 146 (Mir146) Modulates Spermatogonial Differentiation by Retinoic Acid in Mice1

    Science.gov (United States)

    Huszar, Jessica M.; Payne, Christopher J.

    2012-01-01

    ABSTRACT Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3′ untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation. PMID:23221399

  9. Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

    Science.gov (United States)

    Payet, Laurie-Anne; Pineau, Ludovic; Snyder, Ellen C R; Colas, Jenny; Moussa, Ahmed; Vannier, Brigitte; Bigay, Joelle; Clarhaut, Jonathan; Becq, Frédéric; Berjeaud, Jean-Marc; Vandebrouck, Clarisse; Ferreira, Thierry

    2013-12-01

    Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

  10. Salicylic acid promotes seed germination under high salinity by modulating antioxidant activity in Arabidopsis.

    Science.gov (United States)

    Lee, Sangmin; Kim, Sang-Gyu; Park, Chung-Mo

    2010-10-01

    • Findings regarding the role of salicylic acid (SA) in seed germination are somewhat variable, depending on the plant genotypes and experimental conditions used, and thus the molecular mechanisms underlying SA regulation of germination are still unclear. Here, we report that physiological concentrations of SA promote germination under high salinity by modulating antioxidant activity in Arabidopsis. • Germination of SA induction deficient 2 (sid2) seeds was hypersensitive to high salinity. While the inhibitory effect of high salinity was exaggerated in the presence of higher concentrations of SA (> 100 μM), it was significantly reduced in the presence of lower concentrations of SA (salinity, the endogenous contents of H(2) O(2) were elevated in wild-type and sid2 seeds but reduced to original concentrations after treatment with 1 μM SA. • Germination of NahG transgenic plants was influenced to a lesser degree by high salinity (NahG is a bacterial gene encoding salicylate hydroxylase that converts salicylic acid to catechol). We found that catechol, an SA degradation product accumulated in the transgenic plants, acts as an antioxidant that compromises the inhibitory effects of high salinity. • Our observations indicate that, although SA is not essential for germination under normal growth conditions, it plays a promotive role in seed germination under high salinity by reducing oxidative damage.

  11. 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.

  12. 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.

  13. 乳酸菌Enterococcuse faecalis TN-9低温蛋白酶的提纯及性质%Purification and Properties of Cold-Adapted Protease from Lactic Acid Bacterium Enterococcus faecalis TN-9

    Institute of Scientific and Technical Information of China (English)

    袁清珠; 林笃志; 北村良久; 岛田贵志

    2009-01-01

    对产自乳酸菌Enterococcuse faecalis TN-9的蛋白酶,进行了硫酸铵沉淀,DEAE-Sephadex A-25以及DEAE Cellulofine A-500离子交换层析的3步纯化和特性研究.纯化酶Native PAGE显示1条蛋白带.SDS-PAGE和凝胶层析分子量分别为30 ku及69 ku.纯化酶最适作用温度为30℃,最适作用pH为7.5~8.0,在pH 6.0~9.5和45℃以下条件下稳定,在0℃下显示了6.1%的相对活性,60℃以上热处理完全失去酶活.该酶被EDTA-2Na,Hg~(2+)、Cu~(2+)、Ni~(2+)、Ag~(2+)、Co~(2+)及Pepstatin A不完全抑制.Zn~(2+)对蛋白酶具有明显的激活作用.纯化酶作用于偶氮酪蛋白的K_m和V_max分别为0.098%和72 mg/(h·mg).该酶为N末端VGSEVTLKNS的明胶酶(Gelatinase)的一种,性质属于低温蛋白酶.%Protease from lactic acid bacterium Enterococcus faecalis TN-9 was purified with three steps, ammonium sulfate precipitation, DEAE-Sephadex A-25, and DEAE Cellnlofine A-500 ion exchange chromatography and studied its properties. Native PAGE analysis of the purified enzyme showed a single protein band. The molecular weight was 30 ku by SDS-PAGE analysis and 69 ku by gel chromatography analysis respectively. The optimal reaction temperature and pH of the enzyme were at 30 ℃ and 7.5 ~ 8.0 respeetively. It was stable at pH 6.0 ~ 9.5 and 45 ℃. Under 0 ℃ it showed 6.1% of relative activity. Heat treatment above 60 ℃ it totally lost its activity. The enzyme was ineomplete-ly inhibited by EDTA-2Na, Hg~(2+) , Cu~(2+) ,Ni~(2+) , Ag~(2+) , Co~(2+) , and Pepstatin. Zn~(2+) had obvious activation to the pro-tease. K_m and V_(max) of the purified enzyme were 0.098% and 72 mg/(h·mg) respectively. The enzyme was one of gelatinase with N-terminal sequence of VGSEVTLKNS. Its property belonged to cold-adapted protease.

  14. Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?

    Science.gov (United States)

    Silva, Roberta N; Oliveira, Lilian C G; Parise, Carolina B; Oliveira, Juliana R; Severino, Beatrice; Corvino, Angela; di Vaio, Paola; Temussi, Piero A; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Luiz; Juliano, Maria A

    2017-05-01

    Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R(↓)R-ACC and Z-R(↓)R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. 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...

  16. Expression of the cathelicidin LL-37 is modulated by short chain fatty acids in colonocytes: relevance of signalling pathways

    OpenAIRE

    Schauber, J; Svanholm, C; Termén, S; Iffland, K; Menzel, T.; Scheppach, W; Melcher, R.; Agerberth, B.; Lührs, H.; G. H. Gudmundsson

    2003-01-01

    Background and aims: Short chain fatty acids (SCFA) exert profound effects on the colonic mucosa. In particular, SCFA modulate mucosal immune functions. The antimicrobial cathelicidin LL-37 is expressed by colon epithelial cells. In the present study the effect of SCFA on LL-37 expression was investigated.

  17. 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.

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

    Science.gov (United States)

    Schauperl, Michael; Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    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.

  19. 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.

  20. A Culture-Based Method for Determining the Production of Secreted Protease Inhibitors

    Science.gov (United States)

    Quintero, David; Bermudes, David

    2014-01-01

    We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them. PMID:24632514

  1. Chlorogenic acid from honeysuckle improves hepatic lipid dysregulation and modulates hepatic fatty acid composition in rats with chronic endotoxin infusion

    OpenAIRE

    2016-01-01

    Chlorogenic acid as a natural hydroxycinnamic acid has protective effect for liver. Endotoxin induced metabolic disorder, such as lipid dysregulation and hyperlipidemia. In this study, we investigated the effect of chlorogenic acid in rats with chronic endotoxin infusion. The Sprague-Dawley rats with lipid metabolic disorder (LD group) were intraperitoneally injected endotoxin. And the rats of chlorogenic acid-LD group were daily received chlorogenic acid by intragastric administration. In ch...

  2. Accelerated evolution of crotalinae snake venom gland serine proteases.

    Science.gov (United States)

    Deshimaru, M; Ogawa, T; Nakashima, K; Nobuhisa, I; Chijiwa, T; Shimohigashi, Y; Fukumaki, Y; Niwa, M; Yamashina, I; Hattori, S; Ohno, M

    1996-11-11

    Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

  3. Specific Strains of Lactic Acid Bacteria Differentially Modulate the Profile of Adipokines In Vitro

    Science.gov (United States)

    Fabersani, Emanuel; Abeijon-Mukdsi, María Claudia; Ross, Romina; Medina, Roxana; González, Silvia; Gauffin-Cano, Paola

    2017-01-01

    Obesity induces local/systemic inflammation accompanied by increases in macrophage infiltration into adipose tissue and production of inflammatory cytokines, chemokines, and hormones. Previous studies have shown that probiotics could improve the intestinal dysbiosis induced by metabolic diseases such as obesity, diabetes, and metabolic syndrome. Microorganisms could (directly or indirectly) affect adipokine levels due to their capacity to induce translocation of several intestinal microbial antigens into systemic circulation, which could lead to metabolic endotoxemia or produce immunomodulation in different organs. The aim of the present study was to select non-inflammatory lactic acid bacteria (LAB) strains with the capacity to modulate adipokine secretion by the adipose tissue. We wish to elucidate the role of potential probiotic strains in the regulation of the cross talking between immune cells such as macrophages and adipose cells. Mouse macrophage cell line RAW 264.7 was used for evaluating the ability of 14 LAB strains to induce cytokine production. The LAB strains were chosen based on their previously studied beneficial properties in health. Then, in murine adipocyte culture and macrophage–adipocyte coculture, we determined the ability of these strains to induce cytokines and leptin secretion. Tumor necrosis factor alpha, interleukin 6 (IL-6), IL-10, monocyte chemoattractant protein-1, and leptin levels were measured in cell supernatants. We also performed the detection and quantification of leptin receptor (Ob-Rb) expression in macrophage cell lines stimulated by these LAB strains. Differential secretion profile of cytokines in macrophage cells induced by LAB strains was observed. Also, the levels of Ob-Rb expression diverged among different LAB strains. In LAB-stimulated coculture cells (adipocytes and macrophages), we observed differential production of leptin and cytokines. Furthermore, we detected lower production levels in single culture than

  4. Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids.

    Science.gov (United States)

    Belury, M A; Leyton, J; Patrick, K E; Cumberland, A G; Locniskar, M; Fischer, S M

    1991-09-01

    Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.

  5. Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

    DEFF Research Database (Denmark)

    Kromann-Hansen, Tobias; Lund, Ida K; Liu, Zhuo

    2013-01-01

    Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities...... criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics....

  6. Cathepsin proteases in Toxoplasma gondii

    Science.gov (United States)

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  7. Modulation of genotoxicity of oxidative mutagens by glycyrrhizic acid from Glycyrrhiza glabra L.

    Directory of Open Access Journals (Sweden)

    Prabhjit Kaur

    2012-01-01

    Full Text Available Background: The chemopreventive effects of certain phytoconstituents can be exploited for their use as functional foods, dietary supplements and even as drugs. The natural compounds, acting as anti-genotoxic and free radical scavenging compounds, may serve as potent chemopreventive agents. These can inhibit DNA modulatory activities of mutagens and help preventing pathological processes. Objectives: Present study on Glycyrrhiza glabra L., a promising medicinal plant, widely used in traditional medicine, focused on the bioassay-guided fractionation of its extracts for the isolation of certain phytochemicals with anti-genotoxic potential against oxidative mutagens. Materials and Methods: The methanol extract of Glycyrrhiza glabra rhizomes was subjected to column chromatography, and isolated fraction was evaluated for its anti-genotoxic and antioxidant potential using SOS chromotest, Comet assay, and DPPH radical scavenging assay. Results: GLG fraction, which was characterized as Glycyrrhizic acid, inhibited the genotoxicity of oxidative mutagens viz., H 2 O 2 and 4NQOquite efficiently. In SOS chromotest, using E.coli PQ37 tester strain, it inhibited induction factor induced by H 2 O 2 and 4NQO by 75.54% and 71.69% at the concentration of 121.46 μM,respectively. In Comet assay, it reduced the tail moment induced by H 2 O 2 and 4NQO by 70.21% and 69.04%, respectively, at the same concentration in human blood lymphocytes. The isolated fraction also exhibited DPPH free radical scavenging activity and was able to scavenge 85.95% radicals at a concentration of 120 μM. Conclusion: Glycyrrhizic acid is a potential modulator of genotoxins as well as efficient scavenger of free radicals.

  8. 20-Hydroxyeicosatetraenoic acid (20-HETE Modulates Canonical Transient Receptor Potential-6 (TRPC6 Channels in Podocytes

    Directory of Open Access Journals (Sweden)

    Hila Roshanravan

    2016-08-01

    Full Text Available The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE regulates renal function, including changes in glomerular function evoked during tubuloglomerular feedback (TGF. This study describes the cellular actions of 20-HETE on cultured podocytes, assessed by whole-cell recordings from cultured podocytes combined with pharmacological and cell-biological manipulations of cells. Bath superfusion of 20-HETE activates cationic currents that are blocked by the pan-TRP blocker SKF-96365 and by 50 μM La3+, and which are attenuated after siRNA knockdown of TRPC6 subunits. Similar currents are evoked by a membrane-permeable analog of diacylgycerol (OAG, but OAG does not occlude responses to maximally-activating concentrations of 20-HETE (20 μM. Exposure to 20-HETE also increased steady-state surface abundance of TRPC6 subunits in podocytes as assessed by cell-surface biotinylation assays, and increased cytosolic concentrations of reactive oxygen species (ROS. TRPC6 activation by 20-HETE was eliminated in cells pretreated with TEMPOL, a membrane-permeable superoxide dismutase mimic. Activation of TRPC6 by 20-HETE was also blocked when whole-cell recording pipettes contained GDP-βS, indicating a role for either small or heterotrimeric G proteins in the transduction cascade. Responses to 20-HETE were eliminated by siRNA knockdown of podocin, a protein that organizes NADPH oxidase complexes with TRPC6 subunits in this cell type. In summary, modulation of ionic channels in podocytes may contribute to glomerular actions of 20-HETE.

  9. Ascorbic acid modulates cell migration in differentiated HL-60 cells and peripheral blood leukocytes.

    Science.gov (United States)

    Schwager, Joseph; Bompard, Albine; Weber, Peter; Raederstorff, Daniel

    2015-08-01

    The impact of L-ascorbic acid (L-AA) on the chemokinesis (CK) and chemotaxis (CT) of HL-60 cells and polymorphonuclear cells (PMN) was investigated. HL-60 cells were differentiated with DMSO, retinoic acid (RA), vitamin D, or L-AA. Chemokinesis and chemotaxis of differentiated HL-cells were assayed. Vitamin D3-treated HL-60 cells (dHL-60vitD3 cells) and RA-treated cells (dHL-60RA cells) acquired monocyte/macrophage-like and neutrophil-like phenotypes, respectively. DMSO induced the differentiation of an intermediate phenotype (dHL-60DMSO cells), whereas L-AA downregulated neutrophil markers (dHL-60L-AA cells). dHL-60DMSO cells had increased CK and potent CT in gradients of IL-8 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). dHL-60RA cells and dHL-60L-AA cells migrated less toward IL-8 and fMLP; dHL-60vitD3 cells preferably responded to fMLP. L-AA enhanced CK of dHL-60DMSO cells and was a weak chemo-attractant. In human leukocytes, IL-8 and fMLP triggered receptor-mediated chemotaxis. CXCR2 and fMLPR were downregulated by IL-8 and fMLP, respectively. L-AA stimulated chemotaxis although significantly less than IL-8 and fMLP. IL-8 targeted chemotaxis was enhanced both in HL-60 cells and leukocytes when cells were incubated with L-AA. L-AA modulated chemokinesis and had significant chemo-attractant properties, which were independent on fMLP or IL-8 receptors. The results suggest that L-AA improves leukocyte function in innate immune responses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates.

    Science.gov (United States)

    De Meis, Leopoldo; Ketzer, Luisa A; Camacho-Pereira, Juliana; Galina, Antonio

    2012-02-01

    The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.

  11. Vermicompost humic acids modulate the accumulation and metabolism of ROS in rice plants.

    Science.gov (United States)

    García, Andrés Calderín; Santos, Leandro Azevedo; de Souza, Luiz Gilberto Ambrósio; Tavares, Orlando Carlos Huertas; Zonta, Everaldo; Gomes, Ernane Tarcisio Martins; García-Mina, José Maria; Berbara, Ricardo Luis Louro

    2016-03-15

    This work aims to determine the reactive oxygen species (ROS) accumulation, gene expression, anti-oxidant enzyme activity, and derived effects on membrane lipid peroxidation and certain stress markers (proline and malondialdehyde-MDA) in the roots of unstressed and PEG-stressed rice plants associated with vermicompost humic acid (VCHA) application. The results show that the application of VCHA to the roots of unstressed rice plants caused a slight but significant increase in root ROS accumulation and the gene expression and activity of the major anti-oxidant enzymes (superoxide dismutase and peroxidase). This action did not have negative effects on root development, and an increase in both root growth and root proliferation occurred. However, the root proline and MDA concentrations and the root permeability results indicate the development of a type of mild stress associated with VCHA application. When VCHA was applied to PEG-stressed plants, a clear alleviation of the inhibition in root development linked to PEG-mediated osmotic stress was observed. This was associated with a reduction in root ROS production and anti-oxidant enzymatic activity caused by osmotic stress. This alleviation of stress caused by VCHA was also reflected as a reduction in the PEG-mediated concentration of MDA in the root as well as root permeability. In summary, the beneficial action of VCHA on the root development of unstressed or PEG-stressed rice plants clearly involves the modulation of ROS accumulation in roots.

  12. Azithromycin Modulates 3',5'-cyclic Diguanylic Acid Signaling in Pseudomonas aeruginosa.

    Science.gov (United States)

    Kimura, Soichiro; Mori, Nobuaki; Kai, Toshihiro; Ishii, Yoshikazu; Yamaguchi, Keizo; Tateda, Kazuhiro

    2017-08-01

    Macrolides have been reported to exert a variety of effects on both host immunomodulation and repression of bacterial pathogenicity. In this study, we report that the 3',5'-cyclic diguanylic acid (c-di-GMP) signaling system, which regulates virulence in Pseudomonas aeruginosa, is affected by the macrolide azithromycin. Using DNA microarray analysis, we selected a gene encoding PA2567 related to c-di-GMP metabolism that was significantly affected by azithromycin treatment. Expression of the PA2567 gene was significantly repressed by azithromycin in a time- and dose-dependent manner, whereas no difference in PA2567 gene expression was observed in the absence of azithromycin. In-frame deletion of the PA2567 gene affected both virulence factors and the quorum-sensing system, and significantly decreased total bacteria in a mouse pneumonia model compared to the wild-type strain (P < 0.05). These results suggest that macrolides possess the ability to modulate c-di-GMP intracellular signaling in P. aeruginosa. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  13. Dynamics of Ca2+-dependent Cl- channel modulation by niflumic acid in rabbit coronary arterial myocytes.

    Science.gov (United States)

    Ledoux, Jonathan; Greenwood, Iain A; Leblanc, Normand

    2005-01-01

    Calcium-activated chloride channels (Cl(Ca)) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. Niflumic acid (NFA), a potent blocker of Cl(Ca) in vascular myocytes, was shown recently to cause inhibition and paradoxical stimulation of sustained calcium-activated chloride currents [I(Cl(Ca))] in rabbit pulmonary artery myocytes. The aims of the present study were to investigate whether NFA produced a similar dual effect in coronary artery smooth muscle cells and to determine the concentration-dependence and dynamics of such a phenomenon. Sustained I(Cl(Ca)) evoked by intracellular Ca(2+) clamped at 500 nM were dose-dependently inhibited by NFA (IC(50) = 159 microM) and transiently augmented in a concentration-independent manner (10 microM to 1 mM) approximately 2-fold after NFA removal. However, the time to peak and duration of NFA-enhanced I(Cl(Ca)) increased in a concentration-dependent fashion. Moreover, the rate of recovery was reduced by membrane depolarization, suggesting the involvement of a voltage-dependent step in the interaction of NFA, leading to stimulation of I(Cl(Ca)). Computer simulations derived from a kinetic model involving low (K(i) = 1.25 mM) and high (K(i) < 30 microM) affinity sites could reproduce the properties of the NFA-modulated I(Cl(Ca)) fairly well.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 /sup 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 /sup 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.

  15. Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within Medicago sativa nodules.

    Science.gov (United States)

    Bianco, C; Senatore, B; Arbucci, S; Pieraccini, G; Defez, R

    2014-07-01

    To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.

  16. Modulation of Acid Sphingomyelinase in Melanoma Reprogrammes the Tumour Immune Microenvironment

    Directory of Open Access Journals (Sweden)

    Emma Assi

    2015-01-01

    Full Text Available The inflammatory microenvironment induces tumours to acquire an aggressive and immunosuppressive behaviour. Since acid sphingomyelinase (A-SMase downregulation in melanoma was shown to determine a malignant phenotype, we aimed here to elucidate the role of A-SMase in the regulation of tumour immunogenic microenvironment using in vivo melanoma models in which A-SMase was either downregulated or maintained at constitutively high levels. We found high levels of inflammatory factors in low A-SMase expressing tumours, which also displayed an immunosuppressive/protumoural microenvironment: high levels of myeloid-derived suppressor cells (MDSCs and regulatory T lymphocytes (Tregs, as well as low levels of dendritic cells (DCs. In contrast, the restoration of A-SMase in melanoma cells not only reduced tumour growth and immunosuppression, but also induced a high recruitment at tumour site of effector immune cells with an antitumoural function. Indeed, we observed a poor homing of MDSCs and Tregs and the increased recruitment of CD8+ and CD4+ T lymphocytes as well as the infiltration of DCs and CD8+/CD44high T lymphocytes. This study demonstrates that change of A-SMase expression in cancer cells is sufficient per se to tune in vivo melanoma growth and that A-SMase levels modulate immune cells at tumour site. This may be taken into consideration in the setting of therapeutic strategies.

  17. Potential role of salicylic acid in modulating diacylglycerol homeostasis in response to freezing temperatures in Arabidopsis.

    Science.gov (United States)

    Tan, Wei-Juan; Xiao, Shi; Chen, Qin-Fang

    2015-01-01

    In our recent article in Molecular Plant, we reported that 3 lipase-like defense regulators SENESCENCE-ASSOCIATED GENE101 (SAG101), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are involved in the regulation of freezing tolerance in Arabidopsis. The transcripts of SAG101, EDS1 and PAD4 were inducible by cold stress and their knockout or knockdown mutants exhibited enhanced chilling and freezing tolerance in comparison to the wild type. The freezing tolerance phenotype showed in the sag101, eds1 and pad4 mutants was correlated with the transcriptional upregulation of C-REPEAT/DRE BINDING FACTORs (CBFs) and their regulons as well as increased levels of proline. Upon cold exposure, the sag101, eds1 and pad4 mutants showed ameliorated cell death and accumulation of hydrogen peroxide, which were highly induced by freezing stress in the wild-type leaves. Moreover, the contents of salicylic acid (SA) and diacylglycerol (DAG) were significantly decreased in the sag101, eds1 and pad4 mutants compared to the wild type. Taken together, our results suggest that the SAG101, EDS1 and PAD4 are negative regulators in the freezing response and function, at least in part, by modulating the homeostasis of SA and DAG in Arabidopsis.

  18. 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

  19. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

    DEFF Research Database (Denmark)

    Loechel, F; Overgaard, M T; Oxvig, C

    1999-01-01

    , with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition...... of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity...... of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease...

  20. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  1. Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

    OpenAIRE

    Cupp-Enyard, Carrie

    2009-01-01

    The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.

  2. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. 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.

  4. 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; 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.

  5. 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

  6. 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.

  7. Modeling and structural analysis of PA clan serine proteases

    Directory of Open Access Journals (Sweden)

    Laskar Aparna

    2012-05-01

    Full Text Available Abstract Background Serine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques. Results Our predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core

  8. Caffeic acid phenethyl ester modulates aflatoxin B1-induced hepatotoxicity in rats.

    Science.gov (United States)

    Akçam, Mustafa; Artan, Reha; Yilmaz, Aygen; Ozdem, Sebahat; Gelen, Tekinalp; Nazıroğlu, Mustafa

    2013-12-01

    Aflatoxin B1 (AFB1) is the most potent of the mycotoxins and is widely observed in nutrition abnormalities. There are some studies suggesting oxidative stress-induced toxic changes on liver related to AFB1 toxicity. The aim of the present study was to evaluate whether antioxidant caffeic acid phenethyl ester (CAPE) relieves oxidative stress in AFB1-induced liver injury in rat. Twenty-four male rats were equally divided into three groups. The first group was used as a control. The second group received three doses of AFB1. The three doses of CAPE were given to constitute the third group with doses of AFB1. After 10 days of experiment, liver and serum samples were taken from all animals. Serum gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), glutathione s-transferase (GST), nitric oxide (NO) and sulfhydryl values were higher in the AFB1 group than in control, whereas serum GGT, ALP, GST and NO values were decreased by in the AFB1 + CAPE group than in AFB1 group. Liver GST, total oxidant capacity, sulfhydryl, apoptosis index and ischemia-modified albumin values were higher in the AFB1 group than in control, whereas the GST activity and apoptosis index were lower in the AFB1 + CAPE group than in the AFB1 group. There were histopathological degeneration and apoptosis in hepatocytes of AFB1 group. The findings were totally recovered by CAPE administration. In conclusion, we observed that AFB1 caused oxidative and nitrosative hepatoxicity to hepatocytes in the rat. However, CAPE induced protective effects on the AFB1-induced hepatoxicity by modulating free radical production, biochemical values and histopathological alterations.

  9. N-3 fatty acids modulate Th1 and Th2 dichotomy in diabetic pregnancy and macrosomia.

    Science.gov (United States)

    Khan, Naim Akhtar; Yessoufou, Akadiri; Kim, Minji; Hichami, Aziz

    2006-06-01

    We assessed the implication of Th (helper)-cells and the modulation of the Th1/Th2 dichotomy by n-3 polyunsaturated fatty acids (PUFA) in type I diabetic pregnancy (DP) and macrosomia. Female gestant rats fed a standard diet or n-3 PUFA regimen were rendered diabetic by administration of five low doses of streptozotocin. The macrosomic (MAC) offspring were sacrificed at the age of 90 days. The mRNAs of IL-2 and IFN-gamma (Th1 cytokines) and IL-4 (Th2 cytokine) were downregulated in the pancreas and spleen of diabetic pregnant rats. The levels of IL-10 mRNA, another Th2 cytokine, were unchanged in the spleen or upregulated in the pancreas of these animals. Feeding an n-3 PUFA diet to rats with DP upregulated IL-10 mRNA in the pancreas and IL-4 and IL-10 mRNA in the spleen. In MAC offspring, high expression of IL-2 and IFN-gamma mRNA, but not of Th2 cytokines, was observed. The n-3 PUFA diet diminished Th1 mRNA quantities and increased the levels of IL-4, but not of IL-10, mRNA in MAC offspring. Our study shows that DP is associated with a decreased Th1 phenotype and IL-4 mRNA expression in the pancreas and spleen, and an n-3 PUFA diet upregulates Th2 profile. In MAC offspring, the Th1 phenotype is upregulated and an n-3 PUFA diet downregulates this phenomenon.

  10. Krebs cycle intermediates modulate thiobarbituric acid reactive species (TBARS) production in rat brain in vitro.

    Science.gov (United States)

    Puntel, Robson L; Nogueira, Cristina W; Rocha, João B T

    2005-02-01

    The aim of this study was to investigate the effect of Krebs cycle intermediates on basal and quinolinic acid (QA)- or iron-induced TBARS production in brain membranes. Oxaloacetate, citrate, succinate and malate reduced significantly the basal and QA-induced TBARS production. The potency for basal TBARS inhibition was in the order (IC50 is given in parenthesis as mM) citrate (0.37) > oxaloacetate (1.33) = succinate (1.91) > > malate (12.74). alpha-Ketoglutarate caused an increase in TBARS production without modifying the QA-induced TBARS production. Cyanide (CN-) did not modify the basal or QA-induced TBARS production; however, CN- abolished the antioxidant effects of succinate. QA-induced TBARS production was enhanced by iron ions, and abolished by desferrioxamine (DFO). The intermediates used in this study, except for alpha-ketoglutarate, prevented iron-induced TBARS production. Oxaloacetate, citrate, alpha-ketoglutarate and malate, but no succinate and QA, exhibited significantly iron-chelating properties. Only alpha-ketoglutarate and oxaloacetate protected against hydrogen peroxide-induced deoxyribose degradation, while succinate and malate showed a modest effect against Fe2+/H2O2-induced deoxyribose degradation. Using heat-treated preparations citrate, malate and oxaloacetate protected against basal or QA-induced TBARS production, whereas alpha-ketoglutarate induced TBARS production. Succinate did not offer protection against basal or QA-induced TBARS production. These results suggest that oxaloacetate, malate, succinate, and citrate are effective antioxidants against basal and iron or QA-induced TBARS production, while alpha-ketoglutarate stimulates TBARS production. The mechanism through which Krebs cycle intermediates offer protection against TBARS production is distinct depending on the intermediate used. Thus, under pathological conditions such as ischemia, where citrate concentrations vary it can assume an important role as a modulator of oxidative

  11. 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 ...... of EAATs and their intricate transport process, the novel approaches to pharmacological modulation of the transporters that have emerged, and interesting new perspectives in EAAT as drug targets proposed in recent years....

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

    Directory of Open Access Journals (Sweden)

    Pützer Brigitte M

    2010-11-01

    Full Text Available Abstract Background 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. Results 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. Conclusions 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.

  13. Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

    Science.gov (United States)

    Jones, Kristen L G; Holloway, M Katharine; Su, Hua-Poo; Carroll, Steven S; Burlein, Christine; Touch, Sinoeun; DiStefano, Daniel J; Sanchez, Rosa I; Williams, Theresa M; Vacca, Joseph P; Coburn, Craig A

    2010-07-15

    A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

  14. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes.

    Science.gov (United States)

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo

    2013-03-01

    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  15. Purification and characterization of an extracellular protease from Clonostachys rosea

    Institute of Scientific and Technical Information of China (English)

    LI Jun; HUANG Xiao-wei; ZHANG Ke-qin

    2004-01-01

    @@ An extracellular protease from Clonostachys rosea (syn. Gliocladium roseum) was purified to SDSPAGE homogeneity with 14-fold purification by ultrafiltration、 ammonium sulfate precipetation、hydrophobic interaction chromatography and anion exchange chromatography. The molecular weight of the protease was 32 kDa as estimated by SDS-PAGE. The N-terminal sequence of first 10 amino acids was A-T-Q-S-N-A-P-W-G-L. This enzyme exhibited pH and temperature optima of 9-10 and 60℃, respectively, and was stable over a wide range of pH 4-10 and temperature 4-50 ℃. It did not require Ca2+ for activity and thermal stability. Pre-incubation of enzyme with Zn2+ , Cu2+ , Hg2+,Fe3+ inhibited most of the enzyme activity, but Mn2+ increased enzyme activity up to 38%. It remained stable in the presence of Tween20, H2O2, EDTA. The inhibition profile of the enzymes by PMSF, suggested that this purified protease belongs to the serine protease family. The protease could immobilize nematodes (Panagrellus redivirus) in bioassays and hydrolyzed proteins of the purified cuticle.

  16. Microbial aspartic proteases: current and potential applications in industry.

    Science.gov (United States)

    Theron, Louwrens W; Divol, Benoit

    2014-11-01

    Aspartic proteases are a relatively small group of proteolytic enzymes that are active in acidic environments and are found across all forms of life. Certain microorganisms secrete such proteases as virulence agents and/or in order to break down proteins thereby liberating assimilable sources of nitrogen. Some of the earlier applications of these proteolytic enzymes are found in the manufacturing of cheese where they are used as milk-clotting agents. Over the last decade, they have received tremendous research interest because of their involvement in human diseases. Furthermore, there has also been a growing interest on these enzymes for their applications in several other industries. Recent research suggests in particular that they could be used in the wine industry to prevent the formation of protein haze while preserving the wines' organoleptic properties. In this mini-review, the properties and mechanisms of action of aspartic proteases are summarized. Thereafter, a brief overview of the industrial applications of this specific class of proteases is provided. The use of aspartic proteases as alternatives to clarifying agents in various beverage industries is mentioned, and the potential applications in the wine industry are thoroughly discussed.

  17. Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom.

    Science.gov (United States)

    Nikandrov, Nikolai N; Deshimaru, Masanobu; Tani, Ayako; Chijiwa, Takahito; Shibata, Hiroki; Chang, Chang-Chun; Fukumaki, Yasuyuki; Ito, Tatsumi; Ohno, Motonori

    2005-12-15

    Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1,630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k(cat)/K(m) values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

  18. Omega-3 fatty acids and cytochrome P450-derived eicosanoids in cardiovascular diseases: Which actions and interactions modulate hemodynamics?

    Science.gov (United States)

    Bonafini, Sara; Fava, Cristiano

    2017-01-01

    Increasing interest is focused on omega-3 fatty acids (FA) because of their potential beneficial effects, particularly in cardiovascular disease prevention. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two major omega-3 FA, are mainly consumed through diet, particularly from fish and seafood intake, whereas alpha-linolenic acid (ALA) is present in high amounts in leafy green vegetables, nuts and seeds. The hypothesis of a cardiovascular protective action of omega-3 FA derives mainly from observational studies, whereas the evidence from interventional studies is not always consistent. Nonetheless, clinical trials and meta-analyses indicate a positive action, at minimum on blood pressure (BP). Omega-3 FA may act through different biological pathways; however, in our review, we seek to revisit, most notably, the role of their metabolites via cytochrome P450 (CYP450) in hemodynamic modulation. We emphasize that the effect of omega-3 FA may depend on their balance with other dietary compounds, particularly omega-6 FA, which compete for the same pathways, thus modulating the production of metabolites. Furthermore, the biological activity of omega-3 FA might be better explained by the complex balance and interactions between a variety of nutrients and polymorphisms of genes implicated in specific metabolic pathways.

  19. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    OpenAIRE

    Yang, Hyun-Jong; Chung, Young-Bae; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine protea...

  20. 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 p...... a short-term very low-calorie diet (VLCD)....

  1. 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

    investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured...... of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing......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...

  2. Gene expression signature of DMBA-induced hamster buccal pouch carcinomas: modulation by chlorophyllin and ellagic acid.

    Directory of Open Access Journals (Sweden)

    Ramamurthi Vidya Priyadarsini

    Full Text Available Chlorophyllin (CHL, a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA, a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA-induced hamster buccal pouch (HBP carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy.

  3. Gene expression signature of DMBA-induced hamster buccal pouch carcinomas: modulation by chlorophyllin and ellagic acid.

    Science.gov (United States)

    Vidya Priyadarsini, Ramamurthi; Kumar, Neeraj; Khan, Imran; Thiyagarajan, Paranthaman; Kondaiah, Paturu; Nagini, Siddavaram

    2012-01-01

    Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy.

  4. 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.

  5. Alleviation of selenium toxicity in Brassica juncea L.: salicylic acid-mediated modulation in toxicity indicators, stress modulators, and sulfur-related gene transcripts.

    Science.gov (United States)

    Gupta, Shikha; Gupta, Meetu

    2016-11-01

    The present work reveals the response of different doses of selenium (Se) and alleviating effect of salicylic acid (SA) on Se-stressed Brassica juncea seedlings. Selenium, a micronutrient, is essential for both humans and animals but is toxic at higher doses. Its beneficial role for the survival of plants, however, is still debatable. On the other hand, SA, a phenolic compound, is known to have specific responses under environmental stresses. Experiments were conducted using leaves of hydroponically grown seedlings of Pusa bold (PB) variety of B. juncea, treated with different concentrations of Se (50, 150, 300 μM) for 24- and 96-h exposure times. Increasing Se concentrations inhibited growth and, caused lipid peroxidation, concomitantly increased stress modulators (proline, cysteine, SOD, CAT) along with sulfur-related gene transcripts (LAST, APS, APR, GR, OASL, MT-2, PCS) in Brassica seedlings. On the basis of the above studied parameters, maximum inhibition in growth was observed at 300 μM Se after 96-h exposure time. Further, co-application of SA along with 300 μM Se helped to mitigate Se stress, as shown by improved levels of growth parameters, toxicity indicators (chlorophyll, protein, MDA), stress modulators (proline, cysteine, SOD, and CAT), and expression of sulfur-related genes as compared to Se-treated seedlings alone. Altogether, this study revealed that Se + SA combinations improved seedling morphology and were effective in alleviation of Se stress in PB variety of B. juncea.

  6. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

    Science.gov (United States)

    Gualdoni, Guido A; Mayer, Katharina A; Göschl, Lisa; Boucheron, Nicole; Ellmeier, Wilfried; Zlabinger, Gerhard J

    2016-11-01

    T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply and modulates cellular metabolism toward a catabolic state. Although this enzyme is known to be particularly active in regulatory T (Treg) cells, its impact on T helper (Th)-cell differentiation is poorly understood. We investigated the impact of several AMPK activators on Treg-cell differentiation and found that the direct activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), but not the indirect activators metformin and 2-deoxyglucose, strongly enhanced Treg-cell induction by specifically enhancing Treg-cell expansion. Conversely, Th17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased Treg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the dependence of AICAR's effects on metabolic pathways alterations. Translating our findings to an in vivo model, we found that the substance enhanced Treg cell generation on IL-2 complex-induced immune stimulation. We provide a previously unrecognized insight into the delicate interplay between immune cell function and metabolism and delineate a potential novel strategy for metabolism-targeting immunotherapy.-Gualdoni, G. A., Mayer, K. A., Göschl, L., Boucheron, N., Ellmeier, W., Zlabinger, G. J. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

  7. 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.

  8. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  9. Dietary omega-3 fatty acids modulate large-scale systems organization in the rhesus macaque brain.

    Science.gov (United States)

    Grayson, David S; Kroenke, Christopher D; Neuringer, Martha; Fair, Damien A

    2014-02-01

    Omega-3 fatty acids are essential for healthy brain and retinal development and have been implicated in a variety of neurodevelopmental disorders. This study used resting-state functional connectivity MRI to define the large-scale organization of the rhesus macaque brain and changes associated with differences in lifetime ω-3 fatty acid intake. Monkeys fed docosahexaenoic acid, the long-chain ω-3 fatty acid abundant in neural membranes, had cortical modular organization resembling the healthy human brain. In contrast, those with low levels of dietary ω-3 fatty acids had decreased functional connectivity within the early visual pathway and throughout higher-order associational cortex and showed impairment of distributed cortical networks. Our findings illustrate the similarity in modular cortical organization between the healthy human and macaque brain and support the notion that ω-3 fatty acids play a crucial role in developing and/or maintaining distributed, large-scale brain systems, including those essential for normal cognitive function.

  10. 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.

  11. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  12. Acetic acid modulates spike rate and spike latency to salt in peripheral gustatory neurons of rats

    Science.gov (United States)

    Breza, Joseph M.

    2012-01-01

    Sour and salt taste interactions are not well understood in the peripheral gustatory system. Therefore, we investigated the interaction of acetic acid and NaCl on taste processing by rat chorda tympani neurons. We recorded multi-unit responses from the severed chorda tympani nerve (CT) and single-cell responses from intact narrowly tuned and broadly tuned salt-sensitive neurons in the geniculate ganglion simultaneously with stimulus-evoked summated potentials to signal when the stimulus contacted the lingual epithelium. Artificial saliva served as the rinse and solvent for all stimuli [0.3 M NH4Cl, 0.5 M sucrose, 0.1 M NaCl, 0.01 M citric acid, 0.02 M quinine hydrochloride (QHCl), 0.1 M KCl, 0.003–0.1 M acetic acid, and 0.003–0.1 M acetic acid mixed with 0.1 M NaCl]. We used benzamil to assess NaCl responses mediated by the epithelial sodium channel (ENaC). The CT nerve responses to acetic acid/NaCl mixtures were less than those predicted by summing the component responses. Single-unit analyses revealed that acetic acid activated acid-generalist neurons exclusively in a concentration-dependent manner: increasing acid concentration increased response frequency and decreased response latency in a parallel fashion. Acetic acid suppressed NaCl responses in ENaC-dependent NaCl-specialist neurons, whereas acetic acid-NaCl mixtures were additive in acid-generalist neurons. These data suggest that acetic acid attenuates sodium responses in ENaC-expressing-taste cells in contact with NaCl-specialist neurons, whereas acetic acid-NaCl mixtures activate distinct receptor/cellular mechanisms on taste cells in contact with acid-generalist neurons. We speculate that NaCl-specialist neurons are in contact with type I cells, whereas acid-generalist neurons are in contact with type III cells in fungiform taste buds. PMID:22896718

  13. Reversible phenotypic modulation induced by deprivation of exogenous essential fatty acids.

    Science.gov (United States)

    Laposata, M; Minda, M; Capriotti, A M; Hartman, E J; Furth, E E; Iozzo, R V

    1988-12-01

    Essential fatty acid deficiency, produced by deprivation of omega-6 and omega-3 fatty acids, is a condition characterized by renal disease, dermatitis, and infertility. Although many of the biochemical aspects of this disorder have been investigated, little is known about the ultrastructural changes induced by essential fatty acid deficiency. Using a unique fatty acid-deficient cell line (EFD-1), which demonstrates the in vivo fatty acid changes of essential fatty acid deficiency, and the prostaglandin E2-producing mouse fibrosarcoma line from which it was derived (HSDM1C1), we correlated ultrastructural and biochemical changes induced by prolonged deprivation of all exogenous lipids and subsequent repletion of selected essential fatty acids. We found that in cells deprived of all exogenous lipids, there was dilation of rough endoplasmic reticulum and an associated defect in protein secretion; these changes were specifically reversed by arachidonate. There was also an accumulation of secondary lysosomes containing degraded membranes in these cells with an associated increase in phospholipids relative to parent HSDM1C1 cells. Cytoplasmic lipid bodies present in parent cells disappeared, with an associated decrease in triacylglycerol. After just 2 days in lipid-free medium, all these changes were apparent, and prostaglandin E2 production was markedly impaired despite normal amounts of cellular arachidonate. Incubation of EFD-1 cells with arachidonate, the major prostaglandin precursor fatty acid, induced a reversion to the HSDM1C1 phenotype, whereas other fatty acids were totally ineffective. These results indicate changes in fatty acid metabolism in essential fatty acid deficiency are associated with marked alterations in ultrastructure and secretion of protein from cells.

  14. "THE ROLE OF PROSTAGLANDINS AND MAST CELLS IN THE MODULATION OF ACUTE ACID-INDUCED TRACHEAL CONTRACTION IN RAT"

    Directory of Open Access Journals (Sweden)

    M. H. Pipelzadeh

    2004-05-01

    Full Text Available The exact role of the epithelial lining of the trachea in modulating the bronchial tone is controversial. The present study was an attempt to verify the role of both prostaglandins and mast cells in the acute phase of acid inspiration in rat. Four groups (n = 6 of N. Mari rats were employed. The first group was used as placebo control, and normal saline was injected. To the second group hydrochloric acid (25 µl with pH of 1.3 was injected into the trachea through the criothyroid membrane. The third and fourth groups were pretreated for three consecutive days either with indomethacin (10 mg/Kg or nebulized sodium cromoglycate (20 mg/Kg, 1 hour prior to installation of the acid. Three minutes after instillation of acid, the trachea was removed. The tracheal spirals were prepared and immediately suspended in an organ bath containing Tyrode’s solution. Dose response curves to acetylcholine (10-9 to 10-3M were constructed. The results showed that the responses to acetylcholine in the acid treated trachea were significantly (P<0.01 reduced compared with the control saline treated trachea due to acute acid-induced tracheal contraction. Incubation with atropine, induced reduction of baseline tension and reversed the responses to acetylcholine. Both indomethacin and sodium cromoglycate, reversed the responses to acetylcholine and were in similar range as the control trachea. In conclusion, it seems that both prostaglandins and mast cells are important mediators in the acute phase of airway smooth muscle contraction following instillation of acid.

  15. 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.

  16. Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.

    Science.gov (United States)

    Boulware, Kevin T; Jabaiah, Abeer; Daugherty, Patrick S

    2010-06-15

    Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non-optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two-color cellular library of peptide substrates (CLiPS) methodology was developed. Two-color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven-amino-acid substrate, with a strong consensus of EXLYPhiQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven-residue TEV substrate co-evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    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 ({sup 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 ({sup 3}H)diazepam binding are those that are active in displacing ({sup 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.

  18. 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.

  19. Lipoprotein lipase variants interact with polyunsaturated fatty acids to modulate obesity traits in Puerto Ricans

    Science.gov (United States)

    Lipoprotein lipase (LPL) is a candidate gene for obesity based on its role in triglyceride hydrolysis and the partitioning of fatty acids towards storage or oxidation. Whether dietary fatty acids modify LPL associated obesity risk is unknown. We examined five single nucleotide polymorphisms (SNPs) (...

  20. 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 5beta-reductase....

  1. 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 an

  2. 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.

  3. Biotechnology of cold-active proteases.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, Tulasi

    2013-05-03

    The bulk of Earth's biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth's surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  4. Biotechnology of Cold-Active Proteases

    Directory of Open Access Journals (Sweden)

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  5. 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

    The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from...

  6. Antidepressant-like effects of ascorbic acid and ketamine involve modulation of GABAA and GABAB receptors.

    Science.gov (United States)

    Rosa, Priscila B; Neis, Vivian B; Ribeiro, Camille M; Moretti, Morgana; Rodrigues, Ana Lúcia S

    2016-10-01

    It has been suggested that dysregulation of γ-aminobutyric acid (GABA)-mediated neurotransmission is involved in the etiology of major depressive disorder and in the action of the fast-acting antidepressant ketamine. Considering that recent evidence has suggested that ascorbic acid may exert an antidepressant-like effect through mechanisms similar to ketamine, this study evaluated the involvement of GABAA and GABAB receptors in the antidepressant-like effect of ascorbic acid, comparing the results with those obtained with ketamine. To investigate the involvement of GABAA in the antidepressant-like effect of ascorbic acid and ketamine in the tail suspension test (TST), mice were treated with a sub-effective dose of ascorbic acid (0.1mg/kg, po), ketamine (0.1mg/kg, ip) or vehicle and 30minutes later, a sub-effective dose of muscimol (0.1mg/kg, ip, GABAA receptor agonist) or vehicle was administered. In another set of experiments, mice were treated with ascorbic acid (1mg/kg, po, active dose in the TST) or vehicle and 30minutes later, baclofen (1mg/kg, ip, GABAB receptor agonist) was administered. A similar experimental protocol was performed with ketamine (1mg/kg, ip). The administration of muscimol combined with ascorbic acid or ketamine produced a synergistic antidepressant-like effect in the TST. Moreover, the antidepressant-like effects of ascorbic acid and ketamine were abolished by baclofen. There was no alteration in spontaneous locomotion in any experimental group. Results indicate that the anti-immobility effect of ascorbic acid and ketamine in TST may involve an activation of GABAA receptors and a possible inhibition of GABAB receptors. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  7. Single amino acids in sucrose rewards modulate feeding and associative learning in the honeybee.

    Science.gov (United States)

    Simcock, Nicola K; Gray, Helen E; Wright, Geraldine A

    2014-10-01

    Obtaining the correct balance of nutrients requires that the brain integrates information about the body's nutritional state with sensory information from food to guide feeding behaviour. Learning is a mechanism that allows animals to identify cues associated with nutrients so that they can be located quickly when required. Feedback about nutritional state is essential for nutrient balancing and could influence learning. How specific this feedback is to individual nutrients has not often been examined. Here, we tested how the honeybee's nutritional state influenced the likelihood it would feed on and learn sucrose solutions containing single amino acids. Nutritional state was manipulated by pre-feeding bees with either 1M sucrose or 1M sucrose containing 100mM of isoleucine, proline, phenylalanine, or methionine 24h prior to olfactory conditioning of the proboscis extension response. We found that bees pre-fed sucrose solution consumed less of solutions containing amino acids and were also less likely to learn to associate amino acid solutions with odours. Unexpectedly, bees pre-fed solutions containing an amino acid were also less likely to learn to associate odours with sucrose the next day. Furthermore, they consumed more of and were more likely to learn when rewarded with an amino acid solution if they were pre-fed isoleucine and proline. Our data indicate that single amino acids at relatively high concentrations inhibit feeding on sucrose solutions containing them, and they can act as appetitive reinforcers during learning. Our data also suggest that select amino acids interact with mechanisms that signal nutritional sufficiency to reduce hunger. Based on these experiments, we predict that nutrient balancing for essential amino acids during learning requires integration of information about several amino acids experienced simultaneously.

  8. In vitro modulation of caprine monocyte immune functions by ω-3 polyunsaturated fatty acids.

    Science.gov (United States)

    Lecchi, Cristina; Invernizzi, Guido; Agazzi, Alessandro; Ferroni, Mariella; Pisani, Laura Francesca; Savoini, Giovanni; Ceciliani, Fabrizio

    2011-09-01

    The in vitro effects of the ω-3 polyunsaturated fatty acids (PUFAs) eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) on phagocytosis and the extracellular respiratory burst in caprine monocytes were assessed. Blood monocytes incubated with increasing concentrations of EPA or DHA (25-200 μM) demonstrated increased phagocytosis compared to unexposed monocytes. Generation of reactive oxygen species (ROS) was not markedly affected in the presence of EPA and DHA, except at 200 μM, at which concentrations monocyte viability was also reduced.

  9. Omega-3 fatty acids are able to modulate the painful symptoms associated to cyclophosphamide-induced-hemorrhagic cystitis in mice.

    Science.gov (United States)

    Freitas, Raquel D S; Costa, Kesiane M; Nicoletti, Natália F; Kist, Luiza W; Bogo, Maurício R; Campos, Maria M

    2016-01-01

    This study investigated the effects of the long-term dietary fish oil supplementation or the acute administration of the omega-3 fatty acid docosahexaenoic acid (DHA) in the mouse hemorrhagic cystitis (HC) induced by the anticancer drug cyclophosphamide (CYP). HC was induced in mice by a single CYP injection (300mg/kg ip). Animals received four different diets containing 10% and 20% of corn or fish oil, during 21days. Separated groups received DHA by ip (1μmol/kg) or intrathecal (i.t.; 10μg/site) routes, 1h or 15min before CYP. The behavioral tests (spontaneous nociception and mechanical allodynia) were carried out from 1h to 6h following CYP injection. Bladder inflammatory changes, blood cell counts and serum cytokines were evaluated after euthanasia (at 6h). Immunohistochemistry analysis was performed for assessing spinal astrocyte and microglia activation or GPR40/FFAR1 expression. Either fish oil supplementation or DHA treatment (ip and i.t.) markedly prevented visceral pain, without affecting CYP-evoked bladder inflammatory changes. Moreover, systemic DHA significantly prevented the neutrophilia/lymphopenia caused by CYP, whereas this fatty acid did not significantly affect serum cytokines. DHA also modulated the spinal astrocyte activation and the GPR40/FFAR1 expression. The supplementation with fish oil enriched in omega-3 fatty acids or parenteral DHA might be interesting nutritional approaches for cancer patients under chemotherapy schemes with CYP.

  10. Identification and mechanism of 10-carbon fatty acid as modulating ligand of peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Malapaka, Raghu R V; Khoo, Sokkean; Zhang, Jifeng; Choi, Jang H; Zhou, X Edward; Xu, Yong; Gong, Yinhan; Li, Jun; Yong, Eu-Leong; Chalmers, Michael J; Chang, Lin; Resau, James H; Griffin, Patrick R; Chen, Y Eugene; Xu, H Eric

    2012-01-02

    Peroxisome proliferator-activated receptors (PPARα, -β/δ, and -γ) are a subfamily of nuclear receptors that plays key roles in glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione class of antidiabetic drugs that has many side effects. PPARγ is also activated by long chain unsaturated or oxidized/nitrated fatty acids, but its relationship with the medium chain fatty acids remains unclear even though the medium chain triglyceride oils have been used to control weight gain and glycemic index. Here, we show that decanoic acid (DA), a 10-carbon fatty acid and a major component of medium chain triglyceride oils, is a direct ligand of PPARγ. DA binds and partially activates PPARγ without leading to adipogenesis. Crystal structure reveals that DA occupies a novel binding site and only partially stabilizes the AF-2 helix. DA also binds weakly to PPARα and PPARβ/δ. Treatments with DA and its triglyceride form improve glucose sensitivity and lipid profiles without weight gain in diabetic mice. Together, these results suggest that DA is a modulating ligand for PPARs, and the structure can aid in designing better and safer PPARγ-based drugs.

  11. Cystatin protease inhibitors and immune functions.

    Science.gov (United States)

    Zavasnik-Bergant, Tina

    2008-05-01

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. They are wide spread in all living organisms (mammals, nematodes, arthropods etc.) and are involved in various biological processes where they regulate normal proteolysis and also take part in disease pathology. Many cystatins show changes in expression and/or localization, as well as changes in secretion, following certain stimuli acting on immune cells. In immune cells, cystatins interfere with antigen processing and presentation, phagocytosis, expression of cytokines and nitric oxide and these ways modify the immune response. Further, it has been suggested that cystatin-type molecules secreted from parasites down-modulate the host immune response. Precise understanding of the regulatory roles on proteolytic enzymes of endogenous and exogenous cystatins, such as those from parasites, will provide us with valuable insight into how immune response could be modulated to treat a specific disease. This review covers some specific functions of individual cystatins, with a particular focus on the relevance of cystatins to the immune response.

  12. Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Satyanarayana Bejjani; Raghu Pullakhandam; Ravinder Punjal; K Madhavan Nair

    2007-01-01

    AIM: To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS: Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS: The caco-2 cell ferritin formation was significantly increased (P < 0.001) with FeSO4 (19.3±9.8 ng/mg protein) and pea ferritin (13.9 ± 6.19 ng/mg protein) compared to the blank digest (3.7 ± 1.8 ng/mg protein). Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION: Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.

  13. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    Science.gov (United States)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  14. Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells.

    Science.gov (United States)

    Goncalves, Aurélie; Gleize, Béatrice; Roi, Stéphanie; Nowicki, Marion; Dhaussy, Amélie; Huertas, Alain; Amiot, Marie-Josèphe; Reboul, Emmanuelle

    2013-10-01

    We have recently shown that vitamin D3 (cholecalciferol) absorption is not a simple passive diffusion but involves cholesterol transporters. As free fatty acids (FAs) modulate cholesterol intestinal absorption and metabolism, we hypothesized that FAs may also interact with vitamin D absorption. Effects of FAs were evaluated at different levels of cholecalciferol intestinal absorption. First, the physicochemical properties of micelles formed with different FAs were analyzed. The micelles were then administered to human Caco-2 cells in culture to evaluate FA effects on (i) cholecalciferol uptake and basolateral efflux and (ii) the regulation of genes coding proteins involved in lipid absorption process. Micellar electric charge was correlated with both FA chain length and degree of unsaturation. Long-chain FAs at 500 μM in mixed micelles decreased cholecalciferol uptake in Caco-2 cells. This decrease was annihilated as soon as the long-chain FAs were mixed with other FAs. Oleic acid significantly improved cholecalciferol basolateral efflux compared to other FAs. These results were partly explained by a modulation of genes coding for lipid transport proteins such as Niemann-pick C1-like 1 and scavenger receptor class B type I. The data reported here show for the first time that FAs can interact with cholecalciferol intestinal absorption at different key steps of the absorption process. Cholecalciferol intestinal absorption may thus be optimized according to oil FA composition.

  15. Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function.

    Science.gov (United States)

    Matott, Michael P; Ruyle, Brian C; Hasser, Eileen M; Kline, David D

    2016-03-01

    The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade withdl-threo-β-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function.

  16. Conjugated linoleic acid (CLA: effect modulation of body composition and lipid profile

    Directory of Open Access Journals (Sweden)

    A. Baddini Feitoza

    Full Text Available Conjugated linoleic acid (CLA refers to a family of polyunsaturated fatty acids, being represented by a group of isomers of linoleic acid called conjugated for having a double bound after a simple bound. Among its isomers, trans-10,cis-12 and cis-9, cis-12 CLA stand out. These isomers can lead to different effects on the body: anticarcinogenic, antidiabetogenic, antiatherogenesis and positive body composition alteration. The objective of this review is to describe their mechanisms of action, effects on body composition, on plasmatic lipoproteins and supplementation. Studies about CLA supplementation show its capacity of reducing fat percentage, body mass and of promoting an improvement in lipid metabolism. One of the adverse effects attributed to one of the isomers is insulin resistance by body fat redistribution. Limitations in the scientific models used in CLA researches make impossible to draw conclusions about the action of this fatty acid on human metabolism.

  17. Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases

    Science.gov (United States)

    Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

    2017-01-01

    Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus, a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases. PMID:28529927

  18. C1A cysteine protease-cystatin interactions in leaf senescence.

    Science.gov (United States)

    Díaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; González-Melendi, Pablo; Martínez, Manuel; Díaz, Isabel

    2014-07-01

    Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

  19. Structural Basis for Specific Recognition of Substrates by Sapovirus Protease

    Directory of Open Access Journals (Sweden)

    Masaru eYokoyama

    2012-09-01

    Full Text Available Sapovirus (SaV protease catalyzes cleavage of the peptide bonds at six sites of a viral polyprotein for the viral replication and maturation. However, the mechanisms by which the protease recognizes the distinct sequences of the six cleavage sites remain poorly understood. Here we examined this issue by computational and experimental approaches. A structural modeling and docking study disclosed two small clefts on the SaV protease cavity that allow the stable and functional binding of substrates to the catalytic cavity via aromatic stacking and electrostatic interactions. An information entropy study and a site-directed mutagenesis study consistently suggested variability of the two clefts under functional constraints. Using this information, we identified three chemical compounds that had structural and spatial features resembling those of the substrate amino acid residues bound to the two clefts and that exhibited an inhibitory effect on SaV protease in vitro. These results suggest that the two clefts provide structural base points to realize the functional binding of various substrates.

  20. THE USE OF DIFFERENT PROTEASES TO HYDROLYZE GLIADINS

    Directory of Open Access Journals (Sweden)

    Peter Socha

    2015-02-01

    Full Text Available Gliadins represent alcohol-soluble fraction of wheat storage proteins which is responsible for development of celiac disease. The only and effective treatment for celiac disease is strict adherence to a gluten-free diet excluding any food made with wheat, as well as rye, barley and possibly oat flour. Enzymatic modification of wheat gliadins seems to be an alternative method for decreasing of celiac activity. The aim of our study was a trial of enzymatic modification of wheat gliadins using fungal (Aspergillus sp., Aspergillus oryzae, Aspergillus niger and bacterial (Bacillus licheniformis, Bacillus stearothermophilus, Bacillus thermoproteolyticus, Streptomyces griseus proteases. The reaction was performed up to 60 min, stopped by addition of appropriate synthetic inhibitor and products of limited proteolysis were analyzed by SDS-PAGE method. From fungal proteases most effective proteolytic activity was observed using acid proteinase from A. niger since wheat gliadins and low molecular weight peptides were completely degraded. Bacterial proteases form B. licheniformis and B. thermoproteolyticus acted very effective and as the result of hydrolysis, the products of lower molecular weight (<15 kDa occurred. Most of the wheat gliadins were susceptible to proteolysis by examined bacterial enzymes (exception were protease from B. stearothermophilus and S. griseus. Although wheat gliadins are susceptible to enzymatic degradation, further analysis (e.g. immunochemical or mass spectrometry are desirable to confirm if the products of proteolysis have lost or at least partially decrease their celiac activity.

  1. Factor VII activating protease. Single nucleotide polymorphisms light the way.

    Science.gov (United States)

    Kanse, S M; Etscheid, M

    2011-08-01

    Factor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

  2. 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...

  3. Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle.

    Science.gov (United States)

    Kirkup, A J; Edwards, G; Green, M E; Miller, M; Walker, S D; Weston, A H

    1996-12-12

    The effects of niflumic acid on whole-cell membrane currents and mechanical activity were examined in the rat portal vein. In freshly dispersed portal vein cells clamped at -60 mV in caesium (Cs+)-containing solutions, niflumic acid (1-100 microM) inhibited calcium (Ca2+)-activated chloride currents (IC1(Ca)) induced by caffeine (10 mM) and by noradrenaline (10 microM). In a potassium (K+)-containing solution and at a holding potential of - 10 mV, niflumic acid (10-100 microM) induced an outward K+ current (IK(ATP)) which was sensitive to glibenclamide (10-30 microM). At concentrations < 30 microM and at a holding potential of -2 mV, niflumic acid had no effect on the magnitude of the caffeine- or noradrenaline-stimulated current (IBK(Ca)) carried by the large conductance, Ca(2+)-sensitive K+ channel (BKCa). However, at a concentration of 100 microM, niflumic acid significantly inhibited IBK(Ca)) evoked by caffeine (10 mM) but not by NS1619 (1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3 H) benzimidazolone; 20 microM). In Cs(+)-containing solutions, niflumic acid (10-100 microM) did not inhibit voltage-sensitive Ca2+ currents. In intact portal veins, niflumic acid (1-300 microM) inhibited spontaneous mechanical activity, an action which was partially antagonised by glibenclamide (1-10 microM), and contractions produced by noradrenaline (10 microM), an effect which was glibenclamide-insensitive. It is concluded that inhibition of ICl(Ca) and stimulation of IK(ATP) both contribute to the mechano-inhibitory actions of niflumic acid in the rat portal vein.

  4. Towards bridging the gap between acid-base transporters and neuronal excitability modulation.

    Science.gov (United States)

    Liu, Ying; Chen, Li-Ming

    2014-01-01

    pH homeostasis is a fundamental regulator of the function of the central nervous system. Dysfunction of acid-base transporters often results in disturbance of neuronal excitability. In a latest issue of Journal of Neuroscience, Jones et al. report that increasing intracellular bicarbonate concentration substantially stimulates the excitability of pyramidal neurons from mouse hippocampus by inhibiting KCNQ potassium channel. The finding shed important new light in understanding the molecular mechanism underlying the regulation of neuronal excitability by acid-base transporters.

  5. Purification and characterization of a new metallo-neutral protease for beer brewing from Bacillus amyloliquefaciens SYB-001.

    Science.gov (United States)

    Wang, Jinjing; Xu, Ailan; Wan, Yansong; Li, Qi

    2013-08-01

    The increased additive amount of adjuncts in the raw materials of Chinese beer requires the usage of protease to release more water-soluble proteins. Here, a metallo-neutral protease suited for brewing industry was purified from Bacillus amyloliquefaciens SYB-001. A 5.6-fold purification of the neutral protease was achieved with a 4-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel-filtration chromatography. The molecular mass of the enzyme was estimated to be 36.8 kDa. The protease was active and stable at a wide range of pH from 6.0-10.0 with an optimum at pH 7.0. The highest activity of the purified enzyme was found at 50 °C. The existence of manganese ion would specifically enhance the protease activity. Comparing with other commercial neutral proteases in China, adding the new neutral protease during mashing process would release more amino acids from wort such as aspartic acid, arginine, methione, and histidine, resulting in a better amino acid profile in wort. Moreover, the wort processed with the new neutral protease had a higher α-amino nitrogen concentration, which would ensure a vigorous yeast growth and better flavor. The study of the enzyme could lay a foundation for its industrial application and further research.

  6. 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.

  7. Maternal omega-3 fatty acids and micronutrients modulate fetal lipid metabolism: A review.

    Science.gov (United States)

    Khaire, Amrita A; Kale, Anvita A; Joshi, Sadhana R

    2015-07-01

    It is well established that alterations in the mother's diet or metabolism during pregnancy has long-term adverse effects on the lipid metabolism in the offspring. There is growing interest in the role of specific nutrients especially omega-3 fatty acids in the pathophysiology of lipid disorders. A series of studies carried out in humans and rodents in our department have consistently suggested a link between omega-3 fatty acids especially docosahexaenoic acid and micronutrients (vitamin B12 and folic acid) in the one carbon metabolic cycle and its effect on the fatty acid metabolism, hepatic transcription factors and DNA methylation patterns. However the association of maternal intake or metabolism of these nutrients with fetal lipid metabolism is relatively less explored. In this review, we provide insights into the role of maternal omega-3 fatty acids and vitamin B12 and their influence on fetal lipid metabolism through various mechanisms which influence phosphatidylethanolamine-N-methyltransferase activity, peroxisome proliferator activated receptor, adiponectin signaling pathway and epigenetic process like chromatin methylation. This will help understand the possible mechanisms involved in fetal lipid metabolism and may provide important clues for the prevention of lipid disorders in the offspring.

  8. Modulation of enzymatic activities by n-3 polyunsaturated fatty acids to support cardiovascular health.

    Science.gov (United States)

    Siddiqui, Rafat A; Harvey, Kevin A; Zaloga, Gary P

    2008-07-01

    Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.

  9. Regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by PPAR agonists.

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    Kylie R Dunning

    Full Text Available Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further

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

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    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

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

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    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

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

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    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.

  13. Protease-sensitive synthetic prions.

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    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.

  14. Inhibitors of lysosomal cysteine proteases

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    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  15. Nelfinavir: fourth protease inhibitor approved.

    Science.gov (United States)

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  16. Oxidative modulation of transient potassium current by arachidonic acid in brain central neurons

    OpenAIRE

    Angelova, Plamena

    2007-01-01

    Der neuronale Zelluntergang bei einer Vielzahl von Krankheiten des ZNS, wie z.B. Morbus Alzheimer (AD) und Temporallappenepilepsie (TLE), wird mit oxidativem Stress sowie Fehlfunktionen von Kaliumkanälen in Verbindung gebracht. In dieser Studie soll die selektive neuronale Sensitivität auf oxidativen Stress durch die Messung der oxidativen Modulation von Kaliumströmen untersucht werden. Dabei werden sternförmige Neuronen der zweiten Schicht des entorhinalen Kortex (EC) (bei AD bereits früh ge...

  17. Protease gene families in Populus and Arabidopsis

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    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background 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. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

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

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    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.

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

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    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

  20. Modulation of visceral fat adipokine secretion by dietary fatty acids and ensuing changes in skeletal muscle inflammation.

    Science.gov (United States)

    Tishinsky, Justine M; De Boer, Anna A; Dyck, David J; Robinson, Lindsay E

    2014-01-01

    Given the link between obesity and insulin resistance, the role of adipose-derived factors in communicating with skeletal muscle to affect its function is important. We sought to determine if high fat diets modulate visceral adipose tissue (VAT) adipokines with subsequent effects on skeletal muscle inflammation and insulin sensitivity. Rats were fed (i) low fat (LF), (ii) high saturated fatty acid (SFA), or (iii) high SFA with n-3 polyunsaturated fatty acid (SFA/n-3 PUFA) diets for 4 weeks. VAT-derived adipokines were measured in adipose conditioned medium (ACM) after 72 h. Next, skeletal muscles from LF-fed rats were incubated for 8 h in (i) control buffer (CON), (ii) CON with 2 mmol·L(-1) palmitate (PALM, positive control), (iii) ACM from LF, (iv) ACM from SFA, or (v) ACM from SFA/n-3 PUFA. ACM from rats fed SFA and SFA/n-3 PUFA had increased (P ≤ 0.05) interleukin-6 (IL-6) (+31%) and monocyte chemoattractant protein-1 (MCP-1) (+30%). Adiponectin was decreased (-29%, P ≤ 0.05) in ACM from SFA, and this was prevented in SFA/n-3 PUFA ACM. Toll-like receptor 4 (TLR4) gene expression was increased (P ≤ 0.05) in PALM soleus muscle (+356%) and all ACM groups (+175%-191%). MCP-1 gene expression was elevated (P ≤ 0.05) in PALM soleus muscle (+163%) and soleus muscle incubated in ACM from animals fed SFA (+159%) and SFA/n-3 PUFA (+151%). Glucose transport was impaired (P ≤ 0.05) in PALM muscles but preserved in ACM groups. Acute exposure of muscle to fatty acid modulated adipokines affects skeletal muscle inflammatory gene expression but not insulin sensitivity.

  1. 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.

  2. Cytotoxicity of copper(II) complexes of N-salicylidene-L-glutamate: modulation by ascorbic acid.

    Science.gov (United States)

    Paulikova, H; Kadlecikova, E; Suchanova, M; Valkova, Z; Rauko, P; Hudecova, D; Valent, A

    2008-01-01

    Cytotoxic/cytostatic activity of N-salicylidene-L-glutamato diaqua copper(II) complex (CuC) against mice leukemia cells L1210 has been estimated and their bioactivity was enhanced by addition of ascorbic acid. The Cu-complex with isoquinoline ligand (IQ-CuC) had stronger cytostatic effect (IC50 =15.6 microM) than parental complex (CuC) and its cytotoxicity several times increased in the presence of 0.1 mM ascorbic acid (IC50 =1.0 microM). The cytotoxicity has been caused by oxidative stress, enhanced creation of TBARS has been confirmed, and formation of 2',7'-dichlorofluorescein from 2',7'- dichlorodihydrofluorescein has been observed, also. Some hallmarks of apoptotic/necrotic death of L1210 cells have been observed by fluorescent microscopy after dyeing of cell with propidium iodide and Hoechst 33342. In addition, it was confirmed that both complexes in the presence of ascorbic acid cleavaged of pDNA. Although these copper complexes were initially prepared as substances with antioxidant properties we have showed that combined treatment of L1210 cells with IQCuC and ascorbic acid induced strong oxidative stress and death of cells. Our results confirmed that physiological concentration of ascorbic acid increases the cytostatic/cytotoxic efficiency of N-salicylidene-L-glutamato diaqua copper(II) complexes.

  3. Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

    Science.gov (United States)

    Sanchez, Laura M; Cheng, Andrew T; Warner, Christopher J A; Townsley, Loni; Peach, Kelly C; Navarro, Gabriel; Shikuma, Nicholas J; Bray, Walter M; Riener, Romina M; Yildiz, Fitnat H; Linington, Roger G

    2016-01-01

    Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

  4. Listeria ivanovii ATCC 19119 strain behaviour is modulated by iron and acid stress.

    Science.gov (United States)

    Longhi, Catia; Ammendolia, Maria Grazia; Conte, Maria Pia; Seganti, Lucilla; Iosi, Francesca; Superti, Fabiana

    2014-09-01

    It has been suggested that the rarity of human listeriosis due to Listeria ivanovii reflects not only host tropism factors but also the rare occurrence of this species in the environment, compared with Listeria monocytogenes. In the present study we evaluate the effects on the reference strain L. ivanovii ATCC 19119 behaviour of two combined stresses, low iron availability and acid environment, that bacteria can encounter in the passage from saprophytic life to the host. In these conditions, L. ivanovii evidenced a different behaviour compared to L. monocytogenes exposed to similar conditions. L. ivanovii was not able to mount an acid tolerance response (ATR) even if, upon entry into the stationary phase in iron-loaded medium, growth phase-dependent acid resistance (AR) was evidenced. Moreover, bacteria grown in iron excess and acidic pH showed the higher invasion value in Caco-2 cells, even though it was not able to efficiently multiply. On the contrary, low iron and acidic conditions improved invasion ability in amniotic WISH cells.

  5. Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

    Directory of Open Access Journals (Sweden)

    Laura M Sanchez

    Full Text Available Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1 was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

  6. Modulation of antimicrobial host defense peptide gene expression by free fatty acids.

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    Lakshmi T Sunkara

    Full Text Available Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP genes in the chicken (PLoS One 2011, 6: e27225. In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.

  7. Free fatty acids and their esters modulate isothermal crystallization of anhydrous milk fat.

    Science.gov (United States)

    Bayard, Mathilde; Leal-Calderon, Fernando; Cansell, Maud

    2017-03-01

    The effect of free fatty acids with different chain lengths or unsaturation degree on anhydrous milk fat (AMF) crystallization was evaluated. The impact of esterification was also studied using three triglycerides. Melted blends containing the additives at concentrations lower than 12wt.% were quenched at 25°C and isothermal crystallization was monitored by pulsed low-resolution nuclear magnetic resonance. In parallel, polarized light microscopy was used to observe the microstructure. Compounds based on long chain saturated fatty acids, i.e. palmitic, stearic, eicosanoic acids, tripalmitin and tristearin accelerated crystallization. Conversely, propanoic, hexanoic and oleic acids slowed down the process, while triacetin had no impact. Interestingly, above a critical concentration, the addition of palmitic, stearic or eicosanoic acids caused a transition from a one-step to two-step process. Gompertz model was used to fit the experimental data and to assess the influence of the molecular properties of the additives on the kinetic parameters. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Amino acids as central synaptic transmitters or modulators in mammalian thermoregulation

    Energy Technology Data Exchange (ETDEWEB)

    Bligh, J.

    1981-11-01

    Of the amino acids that affect the activity of central neurons, aspartate and glutamate (which exert generally excitatory influences) and glycine, taurine, and ..gamma..-aminobutyric acid (GABA) (which generally exert inhibitory influences) are the strongest neurotransmitter candidates. As with other putative transmitter substances, their effects on body temperature when injected into the cerebral ventricles or the preoptic hypothalamus tend to vary within and between species. These effects are uninterpretable without accompanying information regarding effector activity changes and the influences of dose and ambient temperature. Observations necessary for analysis of apparent action have been made in studies of the effects of intracerebroventricular injections of these amino acids into sheep. Aspartate and glutamate have similar excitatory effects on the pathway from cold sensors, whereas taurine and GABA exert inhibitory influences on the neural pathways that activate both heat production and heat loss effectors. Glycine appears to be without effect.

  9. 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

    2017-03-22

    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-C15:0 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-C16:0, iso-C15:0, and anteiso-C15:0, 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-C15:0 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.

  10. Modulation of fatty acids and hydrocarbons in Anabaena 7120 and its ntcA mutant under calcium.

    Science.gov (United States)

    Singh, Savita; Verma, Ekta; Tiwari, Balkrishna; Niveshika; Mishra, Arun Kumar

    2017-02-01

    Calcium being a signaling molecule and mediator of cell response, we examined the modulation in fatty acid and hydrocarbon profiles of wild type cyanobacterium Anabaena sp. PCC 7120 and its ntcA mutant under the influence of different calcium chloride concentrations (0-10 mM). Dynamic modifications in fatty acid and hydrocarbon profile were evident through GC-FID analysis of extracted lipids. In the wild type, increase in CaCl2 (10 mM) resulted in unsaturation of fatty acids (observed in terms of high MUFA/PUFA ratio) while hydrocarbon production was distinctly high in the mutant strain compared to wild type at all tested concentrations. The synthesis of short chain hydrocarbons (C5-C8) were dominated at inhibitory concentration (10 mM CaCl2) in mutant strain. Results suggest that the increase in MUFA/PUFA ratio at inhibitory concentration in wild type, and higher percentage of hydrocarbons in mutant strain, may be attributed to the survival and acclimation strategies under altered calcium environment. Our results also suggest the involvement of the ntcA gene (master regulator of N2 metabolism) in regulation of carbon metabolism; specifically fatty acid, hydrocarbon, and other metabolic compounds essential for maintenance and sustenance of growth under stress condition. Thus, our study outlines basic acclimation response along with possibilities of production of fatty acid and hydrocarbon derived biofuel and other bioactive compounds in Anabaena sp. PCC 7120 under altered calcium levels which could be of biotechnological interest.

  11. 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.

  12. The roles of cysteine proteases and phytocystatins in development and germination of cereal seeds.

    Science.gov (United States)

    Szewińska, Joanna; Simińska, Joanna; Bielawski, Wiesław

    2016-12-01

    Proteolysis is an important process for development and germination of cereal seeds. Among the many types of proteases identified in plants are the cysteine proteases (CPs) of the papain and legumain families, which play a crucial role in hydrolysing storage proteins during seed germination as well as in processing the precursors of these proteins and the inactive forms of other proteases. Moreover, all of the tissues of cereal seeds undergo progressive degradation via programed cell death, which is integral to their growth. In view of the important roles played by proteases, their uncontrolled activity could be harmful to the development of seeds and young seedlings. Thus, the activities of these enzymes are regulated by intracellular inhibitors called phytocystatins (PhyCys). The phytocystatins inhibit the activity of proteases of the papain family, and the presence of an additional motif in their C-termini allows them to also regulate the activity of members of the legumain family. A balance between the levels of cysteine proteases and phytocystatins is necessary for proper cereal seed development, and this is maintained through the antagonistic activities of gibberellins (GAs) and abscisic acid (ABA), which regulate the expression of the corresponding genes. Transcriptional regulation of cysteine proteases and phytocystatins is determined by cis-acting elements located in the promoters of these genes and by the expression of their corresponding transcription factors (TFs) and the interactions between different TFs. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. 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.

  14. Transient Influx of Nickel in Root Mitochondria Modulates Organic Acid and Reactive Oxygen Species Production in Nickel Hyperaccumulator Alyssum murale*

    Science.gov (United States)

    Agrawal, Bhavana; Czymmek, Kirk J.; Sparks, Donald L.; Bais, Harsh P.

    2013-01-01

    Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation. PMID:23322782

  15. 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.

  16. Transient Influx of nickel in root mitochondria modulates organic acid and reactive oxygen species production in nickel hyperaccumulator Alyssum murale.

    Science.gov (United States)

    Agrawal, Bhavana; Czymmek, Kirk J; Sparks, Donald L; Bais, Harsh P

    2013-03-08

    Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.

  17. 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...... method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.......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...

  18. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.

    Science.gov (United States)

    Sainsbury, Frank; Rhéaume, Ann-Julie; Goulet, Marie-Claire; Vorster, Juan; Michaud, Dominique

    2012-12-01

    Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

  19. Brain-specific modulation of kynurenic acid synthesis in the rat

    DEFF Research Database (Denmark)

    Gramsbergen, J B; Hodgkins, P S; Rassoulpour, A;

    1997-01-01

    This study was designed to investigate modulatory mechanisms that control the synthesis of the neuroprotective endogenous excitatory amino acid receptor antagonist kynurenate. De novo kynurenate formation was examined in vitro using tissue slices from rat brain, liver, and kidney. In slices from ...

  20. Phytochemicals that modulate amino acid and peptide catabolism by caprine rumen microbes

    Science.gov (United States)

    Background: Microbe-derived ionophores and macrolide antibiotics are often added to ruminant diets, and growth promotion and feed efficiency are among the benefits. One mechanism is inhibition of microbes that catabolize amino acids or peptides and produce ammonia. Plants also produce antimicrobial ...

  1. Nonyloxytryptamine Mimics Polysialic Acid and Modulates Neuronal and Glial Functions in Cell Culture

    Science.gov (United States)

    2014-01-01

    Dev University, Amritsar, Punjab, India ‡Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Athens, Greece §DoD...acid, Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis , MO, USA); the PSA mimicking peptide (NTHTDPYIYPID; Mehanna et al. 2009

  2. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation

    Science.gov (United States)

    While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the im...

  3. 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 of the...

  4. Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA)

    DEFF Research Database (Denmark)

    Vollmer, Jörg; Jepsen, Jan Stenvang; Uhlmann, Eugen

    2004-01-01

    Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have...

  5. 蛋白酶对8~21日龄肉仔鸡生长性能、氨基酸表观消化率及肠道组织形态的影响%Effects of Protease on Growth Performance, Amino Acid Apparent Digestibility and Intestinal Morphology of Broilers Aged from 8 to 21 Days

    Institute of Scientific and Technical Information of China (English)

    周梁; 张海军; 王晶; 郑旭阳; 齐广海; 武书庚

    2014-01-01

    本试验旨在研究蛋白酶对8~21日龄肉仔鸡生长性能、氨基酸表观消化率及肠道组织形态的影响。选择7日龄体重相近、健康的爱拔益加( AA)肉仔鸡560只(公母各占1/2),随机分为5个组,每组8个重复,每个重复14只鸡。对照组饲喂玉米-豆粕-肉骨粉型基础饲粮,试验组分别饲喂在基础饲粮中添加0.01%、0.02%、0.04%和0.08%蛋白酶的试验饲粮,试验期14 d。结果表明:1)各蛋白酶组肉仔鸡平均日采食量显著高于对照组(P<0.05),0.02%蛋白酶组肉仔鸡平均日增重显著高于对照组( P<0.05)。2)蛋白酶可二次性提高肉仔鸡粗蛋白质和氨基酸(亮氨酸和异亮氨酸除外)的表观消化率( P<0.05),0.02%蛋白酶组肉仔鸡粗蛋白质表观消化率显著高于对照组( P<0.05),0.01%和0.02%蛋白酶组天冬氨酸、苏氨酸、丝氨酸和丙氨酸的表观消化率均显著高于对照组( P<0.05)。3)随蛋白酶添加水平增加,十二指肠隐窝深度呈显著线性升高(P<0.05),绒毛高度/隐窝深度呈显著线性降低(P<0.05);空肠绒毛高度呈显著二次曲线升高(P<0.05),隐窝深度呈显著线性升高(P<0.05)。由此可见,玉米-豆粕-肉骨粉型饲粮添加0.01%和0.02%蛋白酶能够促进肉仔鸡生长前期的小肠发育,提高粗蛋白质及大部分氨基酸表观消化率,从而改善生长性能。%This experiment was conducted to study the effects of protease on growth performance, amino acid apparent digestibility and intestinal morphology of broilers aged from 8 to 21 days.A total of 560 seven-day-old healthy Arbor Acres broilers with a similar body weight were randomly allocated to 5 groups with 8 replicates per group and 14 chickens per replicate.Broilers in the control group were fed a basal diet, and broilers in the experimental groups

  6. Properties of hemolysin and protease produced by Aeromonas trota.

    Directory of Open Access Journals (Sweden)

    Eizo Takahashi

    Full Text Available We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes, one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively.

  7. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation.

    Science.gov (United States)

    Khairallah, Ramzi J; Khairallah, Maya; Gélinas, Roselle; Bouchard, Bertrand; Young, Martin E; Allen, Bruce G; Lopaschuk, Gary D; Deschepper, Christian F; Des Rosiers, Christine

    2008-08-01

    While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the impact of its chronic activation on cardiac energy metabolism using mice overexpressing a constitutively active cytoplasmic guanylate cyclase (GC(+/0)) in cardiomyocytes. Ex vivo working GC(+/0) heart perfusions with (13)C-labeled substrates revealed an altered pattern of exogenous substrate fuel selection compared to controls, namely a 38+/-9% lower contribution of exogenous fatty acids to acetyl-CoA formation, while that of carbohydrates remains unchanged despite a two-fold increase in glycolysis. The lower contribution of exogenous fatty acids to energy production is not associated with changes in energy demand or supply (contractile function, oxygen consumption, tissue acetyl-CoA or CoA levels, citric acid cycle flux rate) or in the regulation of beta-oxidation (acetyl-CoA carboxylase activity, tissue malonyl-CoA levels). However, GC(+/0) hearts show a two-fold increase in the incorporation of exogenous oleate into triglycerides. Furthermore, the following molecular data are consistent with a concomitant increase in triglyceride hydrolysis: (i) increased abundance of hormone sensitive lipase (HSL) protein (24+/-11%) and mRNA (22+/-4%) as well as (ii) several phosphorylation events related to HSL inhibitory (AMPK) and activation (ERK 1/2) sites, which should contribute to enhance its activity. These changes in exogenous fatty acid trafficking in GC(+/0) hearts appear to be functionally relevant, as demonstrated by their resistance to fasting-induced triglyceride accumulation. While the documented metabolic profile of GC(+/0) mouse hearts is partly reminiscent of hypertrophied hearts, the observed changes in lipid trafficking have not been previously documented, and may

  8. Structure and mechanism of rhomboid protease.

    Science.gov (United States)

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

  9. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  10. 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.

  11. Mass spectrometry-assisted protease substrate screening

    DEFF Research Database (Denmark)

    Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim;

    2007-01-01

    Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown....... Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed...

  12. Vitamin E supplementation modulates the biological effects of omega-3 fatty acids in naturally aged rats.

    Science.gov (United States)

    Narayanankutty, Arunaksharan; Kottekkat, Anagha; Mathew, Shaji E; Illam, Soorya P; Suseela, Indu M; Raghavamenon, Achuthan C

    2017-03-01

    Omega-3 fatty acids are well-known class of nutraceuticals with established health benefits. Recently, the oxidation products of these fatty acids are gaining attention, as they are likely to disturb body redox balance. Therefore, the efficacy of omega-3 fats under conditions of diminished antioxidant status, such as aging, is always a concern. Present study assessed the effects of omega-3 fats (DHA and EPA) together with or without vitamin-E in naturally aged rats. It was found that in omega-3 fats alone consumed rats the lipid profile was improved, while in omega-3 fat with vitamin-E-consumed group (OMVE), the hepato protective and antioxidant properties were pronounced, especially the redox status of brain tissue. It is possible that vitamin-E might have reduced the peroxidation of omega-3 fats, thereby allowing their synergistic effects. Hence, the use of vitamin-E along with omega-3 fat may be beneficial under aged conditions.

  13. 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.

  14. Effect and proposed mechanism of vitamin C modulating amino acid regulation of autophagic proteolysis.

    Science.gov (United States)

    Karim, Md Razaul; Kadowaki, Motoni

    2017-08-10

    Autophagy is an intracellular bulk degradation process, induced under nutrient starvation. Failure of autophagy has been recognized as a contributor to aging and multiple age related neurodegenerative diseases. Improving autophagy is a beneficial anti-aging strategy, however very few physiological regulators have been identified. Here, we demonstrate that vitamin C is a nutritional stimulator of autophagy. Supplementation of fresh hepatocytes with vitamin C increased autophagic proteolysis significantly in the presence of amino acids in a dose- and time-dependent manner, although no effect was observed in the absence of amino acids. In addition, inhibitor studies with 3-methyladenine, chloroquine, leupeptin and β-lactone confirmed that vitamin C is active through the lysosomal autophagy and not the proteasome pathway. Furthermore, the autophagy marker LC3 protein was significantly increased by vitamin C, suggesting its possible site of action is at the formation step. Both the reduced (ascorbic acid, AsA) and oxidized form (dehydroascorbic acid, DHA) of vitamin C exhibited equal enhancing effect, indicating that the effect does not depend on the anti-oxidation functionality of vitamin C. To understand the mechanism, we established that the effective dose (50 μM) was 15× lower than the intracellular content suggesting these would be only a minor influx from the extracellular pool. Moreover, transporter inhibitor studies in an AsA deficient ODS model rat revealed more accurately that the enhancing effect on autophagic proteolysis still existed, even though the intracellular influx of AsA was blocked. Taken together, these results provide evidence that vitamin C can potentially act through extracellular signaling. Copyright © 2017. Published by Elsevier B.V.

  15. Towards bridging the gap between acid-base transporters and neuronal excitability modulation

    OpenAIRE

    2014-01-01

    pH homeostasis is a fundamental regulator of the function of the central nervous system. Dysfunction of acid-base transporters often results in disturbance of neuronal excitability. In a latest issue of Journal of Neuroscience, Jones et al. report that increasing intracellular bicarbonate concentration substantially stimulates the excitability of pyramidal neurons from mouse hippocampus by inhibiting KCNQ potassium channel. The finding shed important new light in understanding the molecular m...

  16. Endogenous auxin and jasmonic acid levels are differentially modulated by abiotic stresses in rice

    Directory of Open Access Journals (Sweden)

    Hao eDu

    2013-10-01

    Full Text Available Abiotic stresses such as drought, salinity, and adverse temperatures are major limiting factors for plant growth and reproduction. Plant responses to these stresses are coordinated by arrays of regulatory networks including the induction of endogenous abscisic acid (ABA, a well documented phytohormone for stress responses. However, whether or how these abiotic stresses affect the endogenous biosynthesis or metabolism of other phytohormones remains largely unknown. Here, we report the changes of endogenous indole-3-acetic acid (IAA and jasmonic acid (JA levels and expression of genes related to the biosynthesis or signalling of these hormones in rice under various abiotic stress conditions. The IAA content was decreased after drought stress, but it was significantly increased under cold and heat stresses. And the auxin-regulated gravitropism of root tip was inhibited by cold stress. Many genes involved in the IAA biosynthesis and signalling were changed in transcript level under these stresses, and the changes were essentially in agreement with the change of endogenous IAA level. Interestingly, the endogenous JA content was increased markedly under drought and cold stresses, but it was reduced by heat stress. Accordingly, many genes involved in JA biosynthesis and signalling were induced by drought and cold treatment but these genes were significantly suppressed by heat stress. We concluded that endogenous levels of IAA and JA were differentially regulated by abiotic stresses in rice, implying diverse roles of these hormones in stress responses.

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

    Directory of Open Access Journals (Sweden)

    Soumita De

    2013-01-01

    Full Text Available Objectives: 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. Materials and Methods: 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. Results: 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. Conclusions: The present study indicates that PBE has promising analgesic activity, worthy of future pharmacological consideration.

  18. 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.

  19. Bile acids regulate intestinal cell proliferation by modulating EGFR and FXR signaling.

    Science.gov (United States)

    Dossa, Avafia Y; Escobar, Oswaldo; Golden, Jamie; Frey, Mark R; Ford, Henri R; Gayer, Christopher P

    2016-01-15

    Bile acids (BAs) are synthesized in the liver and secreted into the intestine. In the lumen, enteric bacteria metabolize BAs from conjugated, primary forms into more toxic unconjugated, secondary metabolites. Secondary BAs can be injurious to the intestine and may contribute to disease. The epidermal growth factor receptor (EGFR) and the nuclear farnesoid X receptor (FXR) are known to interact with BAs. In this study we examined the effects of BAs on intestinal epithelial cell proliferation and investigated the possible roles for EGFR and FXR in these effects. We report that taurine-conjugated cholic acid (TCA) induced proliferation, while its unconjugated secondary counterpart deoxycholic acid (DCA) inhibited proliferation. TCA stimulated phosphorylation of Src, EGFR, and ERK 1/2. Pharmacological blockade of any of these pathways or genetic ablation of EGFR abrogated TCA-stimulated proliferation. Interestingly, Src or EGFR inhibitors eliminated TCA-induced phosphorylation of both molecules, suggesting that their activation is interdependent. In contrast to TCA, DCA exposure diminished EGFR phosphorylation, and pharmacological or siRNA blockade of FXR abolished DCA-induced inhibition of proliferation. Taken together, these results suggest that TCA induces intestinal cell proliferation via Src, EGFR, and ERK activation. In contrast, DCA inhibits proliferation via an FXR-dependent mechanism that may include downstream inactivation of the EGFR/Src/ERK pathway. Since elevated secondary BA levels are the result of specific bacterial modification, this may provide a mechanism through which an altered microbiota contributes to normal or abnormal intestinal epithelial cell proliferation.

  20. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Science.gov (United States)

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  1. Protease activity in cockroach and basidiomycete allergen extracts.

    Science.gov (United States)

    Wongtim, S; Lehrer, S B; Salvaggio, J E; Horner, W E

    1993-01-01

    Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 248 (Trypsin, 1.0 U), and 533 micrograms/ml (Pronase, 0.5 U) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 micrograms/ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 micrograms/ml (0.9 TEU/mg) and Pleurotus cap extract released 112 micrograms/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocybe cubensis and spores of Psilocybe and the puffball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@1 mM). Loss of allergen activity as determined by RAST inhibition and immunoprinting correlated with protease activity. Thus, in the preparation and handling of allergen extracts, one should employ conditions that minimize proteolysis.

  2. Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.

    Science.gov (United States)

    Ogino, Hiroyasu; Tsuchiyama, Shotaro; Yasuda, Masahiro; Doukyu, Noriyuki

    2010-03-01

    The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

  3. Stabilities and conformational transitions of various proteases in the presence of an organic solvent.

    Science.gov (United States)

    Ogino, Hiroyasu; Gemba, Yuichi; Yutori, Yoshikazu; Doukyu, Noriyuki; Ishimi, Kosaku; Ishikawa, Haruo

    2007-01-01

    The half-life of the activity of the PST-01 protease that was secreted by organic solvent-tolerant Pseudomonas aeruginosa PST-01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST-01 protease, alpha-chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST-01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.

  4. 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.

  5. Naringin ameliorates acetic acid induced colitis through modulation of endogenous oxido-nitrosative balance and DNA damage in rats

    Science.gov (United States)

    Kumar, Venkatashivam Shiva; Rajmane, Anuchandra Ramchandra; Adil, Mohammad; Kandhare, Amit Dattatraya; Ghosh, Pinaki; Bodhankar, Subhash Laxman

    2014-01-01

    The aim of this study was to evaluate the effect of naringin on experimentally induced inflammatory bowel disease in rats. Naringin (20, 40 and 80 mg/kg) was given orally for 7 days to Wistar rats before induction of colitis by intrarectal instillation of 2 mL of 4% (v/v) acetic acid solution. The degree of colonic mucosal damage was analyzed by examining mucosal damage, ulcer area, ulcer index and stool consistency. Intrarectal administration of 4% acetic acid resulted in significant modulation of serum alkaline phosphatase, lactate dehydrogenase, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) content along with colonic nitric oxide (NO), xanthine oxidase (XO) level and protein carbonyl content in the colonic tissue as well as in blood. Naringin (40 and 80 mg/kg) exerted a dose dependent (P < 0.05) ameliorative effect, as it significantly increased hematological parameter as well as colonic SOD and GSH. There was a significant (P < 0.05) and dose dependant inhibition of macroscopical score, ulcer area along with colonic MDA, MPO activity by the 7 days of pretreatment of naringin (40 and 80 mg/kg). Biochemical studies revealed a significant (P < 0.05) dose dependant inhibition in serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) levels by pretreatment of naringin. Increased levels of colonic NO, XO, protein carbonyl content and DNA damage were also significantly decreased by naringin pretreatment. The findings of the present investigation propose that naringin has an anti-inflammatory, anti-oxidant and anti-apoptotic potential effect at colorectal sites as it modulates the production and expression of oxidative mediators such as MDA, MPO, NO and XO, thus reducing DNA damage. PMID:24683411

  6. The Arabidopsis small GTPase AtRAC7/ROP9 is a modulator of auxin and abscisic acid signalling

    Science.gov (United States)

    Cheung, Alice Y.

    2013-01-01

    Rac-like GTPases or Rho-related GTPases from plants (RAC/ROPs) are important components of hormone signalling pathways in plants. Based on phylogeny, several groups can be distinguished, and the underlying premise is that members of different groups perform distinct functions in the plant. AtRAC7/ROP9 is phylogenetically unique among 11 Arabidopsis RAC/ROPs, and here it was shown that it functions as a modulator of auxin and abscisic acid (ABA) signalling, a dual role not previously assigned to these small GTPases. Plants with reduced levels of AtRAC7/ROP9 had increased sensitivity to auxin and were less sensitive to ABA. On the other hand, overexpressing AtRAC7/ROP9 activated ABA-induced gene expression but repressed auxin-induced gene expression. In addition, both hormones regulated the activity of the AtRAC7/ROP9 promoter, suggesting a feedback mechanism to modulate the signalling output from the AtRAC7/ROP9-controlled molecular switch. High levels of AtRAC7/ROP9 were detected specifically in embryos and lateral roots, underscoring the important role of this protein during embryo development and lateral root formation. These results place AtRAC7/ROP9 as an important signal transducer in recently described pathways that integrate auxin and ABA signalling in the plant. PMID:23918972

  7. 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.

  8. Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP).

    Science.gov (United States)

    Schubert, Thomas; Schlegel, Jacqueline; Schmid, Rainer; Opolka, Alfred; Grassel, Susanne; Humphries, Martin; Bosserhoff, Anja-Katrin

    2010-03-31

    Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

  9. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.

    Science.gov (United States)

    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang

    2016-04-01

    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.

  10. Enzyme histochemical studies of membrane proteases in rat subfornical organ.

    Science.gov (United States)

    De Bault, L E; Mitro, A

    1994-12-01

    Localization of membrane proteases glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transpeptidase (gamma-GTP) were studied in vessels of the rat subfornical organ (SFO), ependyma which cover the surface of the SFO, and adjacent brain structures. Results of enzyme histochemical reactions showed strong activity for EAP, mAAP, and gamma-GTP, but absence of DPP IV in microvessels of SFO. The ependyma which cover the SFO was positive for gamma-GTP, but negative for other studied proteases. Our results showed that the spectrum of enzymes in the majority of the vessels of SFO is similar to that of the microvessels of the adjacent brain tissue which were positive for EAP, mAAP, and gamma-GTP, but negative for DPP IV. The relative intensity of the enzyme reactions in vessels varied from central to lateral locations in the SFO and the adjacent brain tissue. There was also a difference in the relative reaction intensity from one enzyme to the other. The presence and heterogeneous distribution of the enzymes are consistent with the hypothesis that membrane proteases of the microvascular endothelium constitute an enzyme-barrier between blood and parenchyma of the SFO and between blood and brain tissue, and may be involved in metabolism or modulation of various peptides when they contact the plasma membrane of the endothelial cells of the vessels.

  11. Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis.

    Science.gov (United States)

    Hernández-Romano, Pablo; Hernández, Roberto; Arroyo, Rossana; Alderete, John F; López-Villaseñor, Imelda

    2010-09-01

    Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (approximately 82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

  12. Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Walmod, P S; Skladchikova, G; Kawa, A

    1999-01-01

    The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology...... of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased...

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

    OpenAIRE

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

    1992-01-01

    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 ...

  14. Processing and targeting of the thiol protease, aleurain

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1989-01-01

    We have identified a cDNA clone from barley aleurone mRNA that encodes a protein with unusual homologies: the C-terminal portion, about 270 amino acids, is 65% identical to the mammalian thiol protease, cathepsin H. This degree of sequence conservation indicates that the enzyme must have some specific function in both plants and mammals that cannot tolerate further divergence. The N-terminal 1/3 of the protein, about 140 amino acids, has no detectable homologies to other known protein sequences; its function is unknown. In aleurone tissue, the mRNA level is increased by gibberellic acid and decreased by abscisic acid, but is expressed apparently constitutively at high levels in leaf and root tissues. The amino acid sequence and cathepsin H homology suggest that the protein will be both secreted into the endoplasmic reticulum and glycosylated. Using our cDNA clone in a bacterial expression system, we have made a fusion protein containing the protease domain of aleurain, and have used it to raise specific antisera in rabbits. These antibodies identify a 32 kd protein in extracts of aleurone layers that is induced with GA treatment but not secreted; a similarly sized protein is specifically identified in extracts of leaf tissue. Experiments are underway to characterize the pattern of expression in different tissues, to identify the subcellular locations of the protein, to characterize processing of the precursor to the 32 kd mature form, and to purify the enzyme from barley. 2 figs.

  15. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    Science.gov (United States)

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z

    2010-11-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.

  16. 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

    2012-01-01

    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 (Kd). The Kd 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. PMID:22773878

  17. Modulation of inflammatory responses by diterpene acids from Helianthus annuus L.

    Science.gov (United States)

    Díaz-Viciedo, Rodrigo; Hortelano, Sonsoles; Girón, Natalia; Massó, Jose M; Rodriguez, Benjamín; Villar, Angel; de Las Heras, Beatriz

    2008-05-02

    Fractionation of a petroleum ether extract of Helianthus annuus L. led to the isolation of three diterpene acids: grandiflorolic, kaurenoic and trachylobanoic acids. These compounds were studied for potential anti-inflammatory activity on the generation of inflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. At non-toxic concentrations, these compounds reduced, in a concentration-dependent manner nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor (TNF-alpha) production, as well as expression of inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2). All diterpenoids displayed significant in vivo anti-inflammatory activity and suppressed the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mouse ear edema. In addition, inhibition of myeloperoxidase (MPO) activity, an index of cellular infiltration, was observed. In summary, our results suggest that the inhibition of the expression of NOS-2, COX-2 and the release of inflammatory cytokines, is responsible for the anti-inflammatory effects of the diterpenoids isolated from H. annuus L. which likely contributes to the pharmacological action of sunflower.

  18. Hydrogen peroxide modulates abscisic acid signaling in root growth and development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    BAI Ling; ZHOU Yun; ZHANG XiaoRan; SONG ChunPeng; Gao MingQing

    2007-01-01

    Exogenous abscisic acid (ABA) can inhibit root growth and promote formation of more root hairs in the root tip of Arabidopsis. However, the molecular mechanisms that underlie root ABA signaling are largely unknown. We report here that hydrogen peroxide (H2O2) reduces the root growth of wild type,and the phenotype of H2O2 on the root growth is similar to ABA response. Meanwhile ABA-induced changes in the morphology of root system can be partly reversed by ascorbic acid in wild type and abolished in NADPH oxidase defective mutant atrbohF and atrbohC. Further, ABA can induce H2O2 accumulation in the root cells and enhance transcription level of OXI1, which is necessary for many more AOS-dependent processes such as root hair growth in Arabidopsis. Our results suggest that H2O2 as an important signal molecule is required for the ABA-regulated root growth and development in Arabidopsis.

  19. Sesamin modulates gene expression without corresponding effects on fatty acids in Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Schiller Vestergren, A; Wagner, L; Pickova, J; Rosenlund, G; Kamal-Eldin, A; Trattner, S

    2012-09-01

    This study examined the effects of sesamin inclusion in vegetable oil-based diets fed to Atlantic salmon (Salmo salar L.). The diets used differed in n-6/n-3 fatty acid (FA) ratio (0.5 and 1) and sesamin content (high 5.8 g/kg, low 1.16 g/kg and no sesamin). The oils used in the feeds were a mixture of rapeseed, linseed and palm oil. Fish were fed for 4 months. Fatty acids and expression of hepatic genes involved in transcription, lipid uptake, desaturation, elongation and β-oxidation were measured. No major effects on the percentage of DHA in white muscle, liver triacylglycerol and phospholipid fraction were detected. Genes involved in β-oxidation, elongation and desaturation were affected by sesamin addition. Limited effects were seen on any of the transcription factors tested and no effect was seen on the expression of peroxisome proliferator-activated receptors (PPAR). Expression of both SREBP-1 and SREBP-2 increased with sesamin addition. It was concluded that supplementation of fish feed with a high level of sesamin had a negative effect on the growth rate and live weight and did not alter the proportions of DHA in tissues even though gene expression was affected. Thus, more studies are needed to formulate a diet that would increase the percentage of DHA in fish without negative effects on fish growth.

  20. The effect of dietary modulation of sulfur amino acids on cystathionine β synthase-deficient mice.

    Science.gov (United States)

    Kruger, Warren D; Gupta, Sapna

    2016-01-01

    Cystathionine β synthase (CBS) is a key enzyme in the methionine and cysteine metabolic pathway, acting as a metabolic gatekeeper to regulate the flow of fixed sulfur from methionine to cysteine. Mutations in the CBS gene cause clinical CBS deficiency, a disease characterized by elevated plasma total homocysteine (tHcy) and methionine and decreased plasma cysteine. The treatment goal for CBS-deficient patients is to normalize the metabolic values of these three metabolites using a combination of vitamin therapy and dietary manipulation. To better understand the effectiveness of nutritional treatment strategies, we have performed a series of long-term dietary manipulation studies using our previously developed Tg-I278T Cbs(-/-) mouse model of CBS deficiency and sibling Tg-I278T Cbs(+/-) controls. Tg-I278T Cbs(-/-) mice have undetectable levels of CBS activity, extremely elevated plasma tHcy, modestly elevated plasma methionine, and low plasma cysteine. They exhibit several easily assayable phenotypes, including osteoporosis, loss of fat mass, reduced life span, and facial alopecia. The diets used in these studies differed in the amounts of sulfur amino acids or sulfur amino acid precursors. In this review, we will discuss our findings and their relevance to CBS deficiency and the concept of gene-diet interaction. © 2015 New York Academy of Sciences.

  1. HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids

    Science.gov (United States)

    Su, Pei-Yi; Yang, Ching-Jen; Chu, Tien-Hua; Chang, Chih-Hsu; Chiang, Chiayn; Tang, Fan-Mei; Lee, Chih-Yin; Shih, Chiaho

    2016-01-01

    Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature. PMID:27958343

  2. Isolation of a new quinic acid derivative and its antibacterial modulating activity

    Directory of Open Access Journals (Sweden)

    A.R. Gohari

    2010-03-01

    Full Text Available "nBackground and the purpose of the study: The species Hymenocrater calycinus, belongs to the plant family Lamiaceae and grows wildly in the north-east of Iran. Previously, the antimicrobial activity of the plant extracts was reported. In the present study, the bioactivity-guided fractionation of the methanol extract of H. calycinus and the combination effects of the isolated compound with cell wall active agents against S. aureus and E. coli was investigated. "n "nMethods: Column and thin layer chromatographic methods were used for isolation and purification and spectroscopic data (MS, 1H- and 13C-NMR, HMQC, HMBC and 1H-1H COSY were employed for identification of the compound isolated from the extract. A disk diffusion method was used to determine the antibacterial activity of the isolated compound against S. aureus and E. coli in comparison with 7 different antibiotics.Results: The isolated compound 1 was identified as 3-(3, 4- dihydroxyphenyl lactic acid 2-O-quinic acid. Compound 1 (500 µg/disc enhanced antibacterial effect of ampicillin, ciprofloxacin, vancomycin and cefepime against S. aureus and activated the effects of ampicillin and vancomycin against E. coli. "nConclusion: Results showed that the compound 1 was not active against both tested strains at any concentration below 1 mg/disk, and as a result the enhancing effect of the compound could be due its association with antibiotics.

  3. Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner.

    Science.gov (United States)

    Wong, Scott W; Kwon, Myung-Ja; Choi, Augustine M K; Kim, Hong-Pyo; Nakahira, Kiichi; Hwang, Daniel H

    2009-10-02

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.

  4. Wound Healing Effect of Slightly Acidic Electrolyzed Water on Cutaneous Wounds in Hairless Mice via Immune-Redox Modulation.

    Science.gov (United States)

    You, Hae Sun; Fadriquela, Ailyn; Sajo, Ma Easter Joy; Bajgai, Johny; Ara, Jesmin; Kim, Cheol Su; Kim, Soo-Ki; Oh, Jin Rok; Shim, Kwang Yong; Lim, Hyun Kyo; Lee, Kyu-Jae

    2017-01-01

    Acidic electrolyzed water is an innovative sanitizer having a wide-spectrum of applications in food industry, and healthcare industry but little is known on its effect and mechanism in wound healing. The study was conducted to identify the effect and mechanism of slightly acidic electrolyzed water (SAEW) on cutaneous wounds in hairless mice. SAEW (pH: 5-6.5, oxidation reduction potential: 800 mV, chlorine concentration: 25 ppm) was prepared through electrolysis of water and was applied to the wounds of hairless mice three times a day for seven days. Wound size, immune response and oxidative stress were explored and compared to conventional agents such as Betadine and alcohol. We found that SAEW-treated group showed the highest wound reduction percentage (phealing. In line, SAEW treatment decreased pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, keratinocyte chemoattractant, and tumor necrosis factor-α] in serum. Other hallmarks of wound healing, matrixmetalloproteinases (MMP)1 and MMP9 were also upregulated. Collectively, our study indicates that SAEW is effective in wound healing of hairless mice via immune-redox modulation, and heals better/faster than conventional agents.

  5. Evidence for a role of gibberellins in salicylic acid-modulated early plant responses to abiotic stress in Arabidopsis seeds.

    Science.gov (United States)

    Alonso-Ramírez, Ana; Rodríguez, Dolores; Reyes, David; Jiménez, Jesús Angel; Nicolás, Gregorio; López-Climent, María; Gómez-Cadenas, Aurelio; Nicolás, Carlos

    2009-07-01

    Exogenous application of gibberellic acid (GA(3)) was able to reverse the inhibitory effect of salt, oxidative, and heat stresses in the germination and seedling establishment of Arabidopsis (Arabidopsis thaliana), this effect being accompanied by an increase in salicylic acid (SA) levels, a hormone that in recent years has been implicated in plant responses to abiotic stress. Furthermore, this treatment induced an increase in the expression levels of the isochorismate synthase1 and nonexpressor of PR1 genes, involved in SA biosynthesis and action, respectively. In addition, we proved that transgenic plants overexpressing a gibberellin (GA)-responsive gene from beechnut (Fagus sylvatica), coding for a member of the GA(3) stimulated in Arabidopsis (GASA) family (FsGASA4), showed a reduced GA dependence for growth and improved responses to salt, oxidative, and heat stress at the level of seed germination and seedling establishment. In 35S:FsGASA4 seeds, the improved behavior under abiotic stress was accompanied by an increase in SA endogenous levels. All these data taken together suggest that this GA-responsive gene and exogenous addition of GAs are able to counteract the inhibitory effects of these adverse environmental conditions in seed germination and seedling growth through modulation of SA biosynthesis. Furthermore, this hypothesis is supported by the fact that sid2 mutants, impaired in SA biosynthesis, are more sensitive to salt stress than wild type and are not affected by exogenous application of GA(3).

  6. Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease.

    Science.gov (United States)

    Robel, Ivonne; Gebhardt, Julia; Mesters, Jeroen R; Gorbalenya, Alexander; Coutard, Bruno; Canard, Bruno; Hilgenfeld, Rolf; Rohayem, Jacques

    2008-08-01

    Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2' positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2' positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

  7. Modulating NMDA Receptor Function with D-Amino Acid Oxidase Inhibitors: Understanding Functional Activity in PCP-Treated Mouse Model.

    Science.gov (United States)

    Sershen, Henry; Hashim, Audrey; Dunlop, David S; Suckow, Raymond F; Cooper, Tom B; Javitt, Daniel C

    2016-02-01

    -mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development.

  8. Modulating NMDA Receptor Function with D-Amino Acid Oxidase Inhibitors: Understanding Functional Activity in PCP-Treated Mouse Model

    Science.gov (United States)

    Sershen, Henry; Hashim, Audrey; Dunlop, David S.; Suckow, Raymond F.; Cooper, Tom B.; Javitt, Daniel C.

    2016-01-01

    , arguing against a DAAO-mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development. PMID:26857796

  9. Critical amino acids in syndecan-4 cytoplasmic domain modulation of turkey satellite cell growth and development.

    Science.gov (United States)

    Song, Yan; McFarland, Douglas C; Velleman, Sandra G

    2012-02-01

    Syndecan-4 is composed of a core protein and covalently attached glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein is divided into extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain has two conserved regions and a variable region in the middle. The Ser residue in the conserved region 1 and the Tyr residue in the variable region are important in regulating protein kinase C alpha (PKCα) membrane localization and focal adhesion formation. The objective of the current study was to investigate the role of syndecan-4 Ser and Tyr residues in combination with the GAG and N-glycosylated chains in turkey satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Site-directed mutagenesis was used to generate Ser and Tyr mutants with or without GAG and N-glycosylated chains. The wild type and mutant syndecan-4 constructs were transfected into turkey satellite cells. The over-expression of Ser and Tyr mutants increased cell proliferation and differentiation and decreased membrane localization of PKCα. Furthermore, Ser mutants enhanced cellular responsiveness to FGF2. The results from this study are the first demonstration of a role of syndecan-4 cytoplasmic domain Ser and Tyr residues in regulating satellite cell proliferation, differentiation, and the modulation of cellular responsiveness to FGF2.

  10. Using the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases

    Science.gov (United States)

    Souza, Diorge P.; Souza, Valquiria P.; Ikegami, Cecilia M.; Farah, Chuck S.; Marana, Sandro R.

    2016-01-01

    The active site residues in GH1 β-glycosidases are compartmentalized into 3 functional regions, involved in catalysis or binding of glycone and aglycone motifs from substrate. However, it still remains unclear how residues outside the active site modulate the enzymatic activity. To tackle this question, we solved the crystal structure of the GH1 β-glycosidase from Spodoptera frugiperda (Sfβgly) to systematically map its residue contact network and correlate effects of mutations within and outside the active site. External mutations neighbouring the functional residues involved in catalysis and glycone-binding are deleterious, whereas mutations neighbouring the aglycone-binding site are less detrimental or even beneficial. The large dataset of new and previously characterized Sfβgly mutants supports that external perturbations are coherently transmitted to active site residues possibly through contacts and specifically disturb functional regions they interact to, reproducing the effects observed for direct mutations of functional residues. This allowed us to suggest that positions related to the aglycone-binding site are preferential targets for introduction of mutations aiming to further improve the hydrolytic activity of β–glycosidases. PMID:27936116

  11. 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.

  12. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.

    Science.gov (United States)

    Kuda, Ondrej; Rombaldova, Martina; Janovska, Petra; Flachs, Pavel; Kopecky, Jan

    2016-01-15

    Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA.

  13. 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...

  14. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was

  15. 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.

  16. Cadmium and nickel uptake are differentially modulated by salicylic acid in Matricaria chamomilla plants.

    Science.gov (United States)

    Kovácik, Jozef; Grúz, Jirí; Hedbavny, Josef; Klejdus, Borivoj; Strnad, Miroslav

    2009-10-28

    Chamomile (Matricaria chamomilla) is a widely used medicinal plant which also accumulates heavy metals in its above-ground organs. We investigated the effect of the important plant signaling molecule, salicylic acid (SA), on the accumulation of Ni or Cd, by exposing plants over 7 days to 60 microM solutions of individual heavy metals with or without 50 microM SA. Special emphasis was focused on phenolic metabolism-related parameters, not only because of their importance for growth and stress tolerance but also because phenolics are potent antioxidants in human diet. In combined treatments, SA stimulated an increase in soluble proteins of roots and reduced their water content. SA reduced total Cd in the shoot and increased Ni. Total and "intraroot" Ni decreased in Ni + SA treatment, while in the case of Cd, only "intraroot" content decreased in Cd + SA treatment, being correlated with cell wall-bound phenolic acids and lignin. SA was strongly accumulated in roots from the Ni + SA treatment, being correlated with an increase in hydrogen peroxide. In both Cd + SA and Ni + SA treatments, SA enhanced phenylalanine ammonia-lyase activity and accumulation of total soluble phenols, particularly in the roots. Here, we report for the first time that soluble phenols may be involved in Cd shoot-to-root translocation. In the case of Ni, it seems that phenols serve as a root barrier in order to prevent Ni from reaching the above-ground organs. The effects of SA on phenolic metabolism, and the signaling role of ROS in the accumulation of phenols, are discussed.

  17. Dietary supplementation with 5-aminolevulinic acid modulates growth performance and inflammatory responses in broiler chickens.

    Science.gov (United States)

    Sato, K; Matsushita, K; Takahashi, K; Aoki, M; Fuziwara, J; Miyanari, S; Kamada, T

    2012-07-01

    The objective of this study was to investigate the effect of dietary supplementation with 5-aminolevulinic acid (5-ALA) on the immune system, inflammatory response, and growth performance of broiler chickens. The levels of cluster of differentiation 3 (CD3) mRNA in the spleens of chickens gradually increased with dietary 5-ALA concentration, while the expression levels of interleukin (IL)-2 decreased. Mitogen-induced proliferation of splenic mononuclear cells and blood mononuclear cell phagocytosis in chickens fed 0.001 and 0.01% 5-ALA-supplemented diets were significantly greater than in chickens fed a basal diet (control). Plasma thiobarbituric acid reactive substance (TBARS) concentration gradually increased along with 5-ALA supplement concentration. These results provide the first evidence that the use of dietary 0.001 and 0.01% 5-ALA supplementation induces the T-cell immune system via mild oxidative stress in chickens. Three hours after Escherichia coli lipopolysaccharide-induced immune stimulation, the levels of mRNA encoding pro-inflammatory cytokines, such as IL-6 and tumor necrosis factor-like ligand 1A (TL1A), in chickens fed a 0.001% 5-ALA-supplemented diet were significantly lower than those in chickens exposed to other treatments. The plasma caeruloplasmin concentration in chickens fed a 0.001% 5-ALA-supplemented diet was significantly lower than in controls or in chickens fed diets supplemented with other concentrations of 5-ALA 24 h after injection of LPS. In addition, BW at 21 and 50 d of age was significantly higher in chickens fed a 0.001% 5-ALA-supplemented diet than in control chickens. The findings suggest that supplementation of diets with 0.001% 5-ALA could prevent the catabolic changes induced by immunological stimulation. These results show that 5-ALA might be useful as an immunomodulator to stimulate T-cells via mild oxidative stress in growing broiler chickens, thereby improving the growth performance.

  18. Extracts from Tribulus species may modulate platelet adhesion by interfering with arachidonic acid metabolism.

    Science.gov (United States)

    Olas, Beata; Hamed, Arafa I; Oleszek, Wieslaw; Stochmal, Anna

    2015-01-01

    The present work was designed to study the effects of crude extracts from Tribulus pterocarpus, T. pentandrus and T. parvispinus on selected biological functions of human blood platelets in vitro. Platelet suspensions were pre-incubated with extracts from aerial parts of T. pterocarpus, T. pentandrus and T. parvispinus, at the final concentrations of 0.5, 5 and 50 µg/ml. Then, for platelet activation thrombin, was used. The effects of crude extracts from T. pterocarpus, T. pentandrus and T. parvispinus on adhesion of blood platelets to collagen were determined by method according to Tuszynski and Murphy. Arachidonic acid metabolism was measured by the level of thiobarbituric acid reactive substances (TBARS). In these studies we also compared the action of tested crude plant extracts with the effects of the polyphenolic fraction isolated from aerial parts of T. pterocarpus, which has antiplatelet and antioxidative properties. The performed assays demonstrated that the tested crude extract from T. pterocarpus and the phenolic fraction from T. pterocarpus might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of this tested extract and its phenolic fraction on adhesion of resting platelets and thrombin - stimulated platelets to collagen was found. We also observed that the crude extract from T. pterocarpus, like the polyphenolic fraction from T. pterocarpus reduced TBARS production in blood platelets. In the comparative studies, the tested crude extract from T. pterocarpus was not found to be more effective antiplatelet factor, than the polyphenolic fraction from this plant. The results obtained suggest that T. pterocarpus may be a promising source of natural compounds, valuable in the prevention of the enhanced activity of blood platelets in numerous cardiovascular diseases.

  19. Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids.

    Science.gov (United States)

    Amorini, Angela Maria; Lazzarino, Giacomo; Di Pietro, Valentina; Signoretti, Stefano; Lazzarino, Giuseppe; Belli, Antonio; Tavazzi, Barbara

    2017-03-01

    In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ-aminobutyrate (GABA), tyrosine (Tyr), S-adenosylhomocysteine (SAH), l-cystathionine (l-Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N-acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham-operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long-lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l-Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu-Gln/GABA cycle between neurons and astrocytes, and of the methyl-cycle (demonstrated by decrease in Met, and increase in SAH and l-Cystat), throughout the post-injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism.

  20. Protease-degradable electrospun fibrous hydrogels

    Science.gov (United States)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  1. Chemical Equilibrium, Unit 4: Equilibria in Acid-Base Systems. A Computer-Enriched Module for Introductory Chemistry. Student's Guide and Teacher's Guide.

    Science.gov (United States)

    Settle, Frank A., Jr.

    Presented are the teacher's guide and student materials for one of a series of self-instructional, computer-based learning modules for an introductory, undergraduate chemistry course. The student manual for this acid-base equilibria unit includes objectives, prerequisites, pretest, a discussion of equilibrium constants, and 20 problem sets.…

  2. 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.

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

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    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.

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

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    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

  5. Ahp cyclodepsipeptides: the impact of the Ahp residue on the "canonical inhibition" of S1 serine proteases.

    Science.gov (United States)

    Stolze, Sara C; Meltzer, Michael; Ehrmann, Michael; Kaiser, Markus

    2013-07-22

    S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small-molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small-molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small-molecule inhibitors that simultaneously target the S and S' subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β-hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.

  6. Effects of omega-3 fatty acids on orexigenic and anorexigenic modulators at the onset of anorexia.

    Science.gov (United States)

    Ramos, Eduardo J B; Romanova, Irina V; Suzuki, Susumu; Chen, Chung; Ugrumov, Michael V; Sato, Tomoi; Goncalves, Carolina G; Meguid, Michael M

    2005-06-07

    In cancer anorexia, a decrease in food intake (FI) occurs concomitant with changes in orexigenic peptides such as neuropeptide Y (NPY) and anorexigenic peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) and anorexigenic neurotransmitter serotonin. omega-3 Fatty acid (omega-3FA) inhibits cytokine synthesis, and delays tumor appearance, tumor growth, and onset of anorexia in tumor-bearing rats. We hypothesize that, in cancer anorexia, omega-3FA is associated with quantitative reversal of hypothalamic NPY, alpha-MSH, and serotonin receptor (5-HT(1B)-receptor) enhancing FI. Fischer rats were divided into: MCA tumor bearing fed chow (TB-Chow) or omega-3FA diet (TB-omega-3FA) and controls: non-tumor bearing fed chow (NTB-Chow) or omega-3FA diet (NTB-omega-3FA). Rats were euthanized at anorexia and brains were removed for hypothalamic immunohistochemical study, using NPY, alpha-MSH, and 5-HT(1B)-receptor-specific antibodies and slides assessed by image analysis. Immunostaining specificity was controlled by omission of primary or secondary antibodies and pre-absorption test. At anorexia, FI decreased (P anorexia, concomitant with an increase in FI, were probably mediated by omega-3FA inhibition of tumor-induced cytokines.

  7. Cationic amino acid transporter-2 regulates immunity by modulating arginase activity.

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    Robert W Thompson

    2008-03-01

    Full Text Available Cationic amino acid transporters (CAT are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/- mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/- mice developed stronger IFN-gamma responses, nitric oxide (NO production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/- mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/- mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.

  8. Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis.

    Science.gov (United States)

    Fang, Linchuan; Su, Lingye; Sun, Xiaoming; Li, Xinbo; Sun, Mengxiang; Karungo, Sospeter Karanja; Fang, Shuang; Chu, Jinfang; Li, Shaohua; Xin, Haiping

    2016-04-01

    The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 (-) were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis.

  9. Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis

    Science.gov (United States)

    Fang, Linchuan; Su, Lingye; Sun, Xiaoming; Li, Xinbo; Sun, Mengxiang; Karungo, Sospeter Karanja; Fang, Shuang; Chu, Jinfang; Li, Shaohua; Xin, Haiping

    2016-01-01

    The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 − were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis. PMID:27162276

  10. Targeting prolyl endopeptidase with valproic acid as a potential modulator of neutrophilic inflammation.

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    Mojtaba Abdul Roda

    Full Text Available A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP, has been recently characterized in chronic obstructive pulmonary disease (COPD. This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV-visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders.

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

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    Jason R Gerstner

    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.

  12. Symbionts as major modulators of insect health: lactic acid bacteria and honeybees.

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    Alejandra Vásquez

    Full Text Available Lactic acid bacteria (LAB are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

  13. Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species.

    Science.gov (United States)

    Wu, Jian; Sun, Yuefeng; Zhao, Yannan; Zhang, Jian; Luo, Lilan; Li, Meng; Wang, Jinlong; Yu, Hong; Liu, Guifu; Yang, Liusha; Xiong, Guosheng; Zhou, Jian-Min; Zuo, Jianru; Wang, Yonghong; Li, Jiayang

    2015-05-01

    Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.

  14. Long-time Fulvic Acid Supplementation Modulates Hydroxylysyl Glycosylation of Collagen in Mice

    Institute of Scientific and Technical Information of China (English)

    朱少华; 朱金龙; 肖坚; 任亮; 刘良; 周亦武

    2004-01-01

    In order to investigate the impact of fulvic acid (FA) on the hydroxylysyl glycosylation in collagen bio-synthesis, 40 NMRI mice were divided into two groups (n=20 in each group, consisting 10 females and 10 males). The animal was maintained for two generations by different diets:control group with normal water and food and study group with water containing 30mg/L FA and normal food. The second generation of the animal was slaughtered, and the biochemical parameters of collagen content and the degree of collagen hydroxylysyl glycosylation in skin, rib and tibia were detected by biochemical methods. The mean value of collagen in the study group was increased slightly, and no significant difference between study group and control group was found (P>0.05), but the content of glucose-glactose-hydroxylysine (GGH) was significantly decreased in the study group in comparison with the control group (P<0.01). It was suggested that through the decrease of GGH 30 mg/L FA could inhibit the activity of galactosyl-hydroxylysylglucosyl-transferase and further disturb the post-translational modification of collagen intracellularly.

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

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    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.

  16. Physiological modulation of circulating FGF21: relevance of free fatty acids and insulin.

    Science.gov (United States)

    Mai, Knut; Bobbert, Thomas; Groth, Christian; Assmann, Anke; Meinus, Sabine; Kraatz, Jessica; Andres, Janin; Arafat, Ayman M; Pfeiffer, Andreas F H; Möhlig, Matthias; Spranger, Joachim

    2010-07-01

    Fibroblast growth factor 21 (FGF-21), a novel metabolic factor in obesity and fasting metabolism, has been shown to be regulated by supraphysiological levels of free fatty acids (FFAs) under hyperinsulinemic conditions. Interestingly, it is still unclear whether the observed effects of FFAs on FGF-21 are relevant under physiological conditions, and the relative functions of FFAs and insulin within this context also need to be determined. Fourteen healthy men were studied in a randomized controlled crossover trial (RCT) using lipid heparin infusion (LHI) at a dose inducing physiological elevations of FFAs vs. saline heparin infusion. In a second randomized controlled trial, FGF-21 was analyzed in 14 patients with type 1 diabetes (6 men, 8 women) during continuous insulin supply vs. discontinued insulin infusion and subsequently increased lipolysis and ketosis. Circulating FGF-21 increased during physiologically elevated FFAs induced by LHI, which was accompanied by mild hyperinsulinemia. Interestingly, a mild elevation of FFAs resulting from complete insulin deficiency also increased FGF-21 levels. These results from two independent human RCTs suggest that FFAs increase circulating FGF-21, while insulin is only of minor importance under physiological conditions. This mechanism might explain the apparent paradox of increased FGF-21 levels in obesity, insulin resistance, and starvation.

  17. Cholinergic modulation of non-N-methyl-D-aspartic acid glutamatergic transmission in the chick ventral lateral geniculate nucleus.

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    Guo, J-Z; Sorenson, E M; Chiappinelli, V A

    2010-03-17

    Neurotransmission between glutamatergic terminals of retinal ganglion cells and principal neurons of the ventral lateral geniculate nucleus (LGNv) was examined with patch clamp recordings in chick brain slices during electrical stimulation of the optic tract. Since muscarinic and nicotinic receptors are present in high densities in LGNv, the present study examined possible roles of both receptors in modulating retinogeniculate transmission. During whole-cell recordings from LGNv neurons, acetylcholine (ACh, 100 microM) caused an initial increase in amplitudes of optic tract-evoked non-N-methyl-D-aspartic acid (NMDA) glutamatergic postsynaptic currents (PSCs). This increase was unchanged when 1 microM atropine was present, indicating that this initial enhancement of PSCs was due entirely to activation of nicotinic receptors. However, during washout of ACh the amplitudes of evoked PSCs became significantly decreased by 40.4+/-5.0% for several minutes before recovering to their original amplitudes, an effect blocked by 1 microM atropine. Exogenously applied muscarine (10 microM) markedly depressed optic tract-evoked PSCs, and this decrease in amplitude was blocked by atropine. In a second set of experiments, we examined effects of releasing endogenous ACh prior to optic tract stimulation. This was accomplished by stimulation of the lateral portion of LGNv via a separate conditioning electrode. Following a brief train of low intensity conditioning stimuli, non-NMDA glutamatergic PSCs evoked by optic tract stimulation were potentiated. However, at higher conditioning stimulus intensities the PSCs were markedly decreased compared with control, and this decrease was partially blocked by atropine (1 microM). Neither ACh nor muscarine altered amplitudes of PSCs elicited by exogenously applied glutamate. Muscarine significantly reduced the frequency but not the amplitudes of miniature PSCs, consistent with a presynaptic location for muscarinic receptors mediating these

  18. CHANGES IN CORTICAL KYNURENIC ACID BI-DIRECTIONALLY MODULATE PREFRONTAL GLUTAMATE LEVELS AS ASSESSED BY MICRODIALYSIS AND RAPID ELECTROCHEMISTRY

    Science.gov (United States)

    Konradsson-Geuken, Ȧ.; Gash, C. R.; Wu, H.-Q.; Alexander, K. S.; Pellicciari, R.; Schwarcz, R.; Bruno, J.P.

    2010-01-01

    Using two in vivo methods, microdialysis and rapid in situ electrochemistry, this study examined the modulation of extracellular glutamate levels by endogenously produced kynurenic acid (KYNA) in the prefrontal cortex (PFC) of awake rats. Measured by microdialysis, intraperitoneal (i.p.) administration of KYNA's bioprecursor L-kynurenine dose-dependently elevated extracellular KYNA and reduced extracellular glutamate (nadir after 50 mg/kg kynurenine: 60% decrease from baseline values). This dose-dependent decrease in glutamate levels was also seen using a glutamate-sensitive microelectrode array (MEA) (31% decrease following 50 mg/kg kynurenine). The kynurenine-induced reduction in glutamate was blocked (microdialysis) or attenuated (MEA) by co-administration of galantamine (3 mg/kg, i.p.), a drug that competes with KYNA at an allosteric potentiating site of the α7 nicotinic acetylcholine receptor. In separate experiments, extracellular glutamate levels were measured by MEA following the local perfusion (45 min) of the PFC with kynurenine (2.5 μM) or the selective KYNA biosynthesis inhibitor S-ethylsulfonylbenzoylalanine (S-ESBA; 5 mM). In agreement with previous microdialysis studies, systemic kynurenine application produced a reversible reduction in glutamate (nadir: −29%), whereas perfusion with S-ESBA increased glutamate levels reversibly (maximum: +38%). Collectively, these results demonstrate that fluctuations in the biosynthesis of KYNA in the PFC bi-directionally modulate extracellular glutamate levels, and that qualitatively very similar data are obtained by microdialysis and MEA. Since KYNA levels are elevated in the PFC of individuals with schizophrenia, and since prefrontal glutamatergic and nicotinic transmission mediate cognitive flexibility, normalization of KYNA levels in the PFC may constitute an effective treatment strategy for alleviating cognitive deficits in schizophrenia. PMID:20600676

  19. Conformational study of a putative HLTV-1 retroviral protease inhibitor.

    Science.gov (United States)

    Llido, S; d'Estaintot, B L; Dautant, A; Geoffre, S; Picard, P; Precigoux, G

    1993-05-01

    The crystal structure of prolyl-glutaminyl-valyl-statyl-alanyl-leucine (Pro-Gln-Val-Sta-Ala-Leu, C(32)H(57)N(7)0(9).5H(2)0, M(r) = 683.9 + 90.1), a putative HTLV-1 protease inhibitor based on one of the consensus retroviral protease cleavage sequences, and containing the statine residue [(4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid], has been determined by X-ray diffraction. The same molecule has been modelled in the active site of the HTLV-1 protease and both conformations have been compared. The peptide crystallizes as a pentahydrate in space group P2(1) with a = 10.874(2), b = 9.501(2), c = 21.062(5) A, beta = 103.68 (1) degrees, Z = 2, V= 2114.3 A(3), D(x) = 1.21 g cm(-3), micro = 8.02 cm(-1), T= 293 K, lambda(Cu Kalpha) = 1.5418 A. The structure has been refined to an R value of 0.070 for 2152 observed reflections. The peptide main chain can be described as extended and adopts the usual zigzag conformation from the prolyl to the statyl residue. The main difference in conformation between the individual observed and modelled molecules is located on the Sta, Ala and Leu residues with the main chain of the modelled molecule rotated by about 180 degrees as compared to the observed conformation in the crystal state.

  20. Mast cell proteases as pharmacological targets.

    Science.gov (United States)

    Caughey, George H

    2016-05-05

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  1. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury.

    Science.gov (United States)

    Murohara, T; Guo, J P; Lefer, A M

    1995-09-01

    Polymorphonuclear neutrophils (PMN) play an important role in myocardial ischemia/reperfusion (MI/R) injury; however, the role of neutrophilic proteases is less understood. The effects of a novel serine protease inhibitor (serpin), LEX032, were investigated in a murine model of MI (20 min) and R (24 hr) injury in vivo. LEX032 is a recombinant human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of alpha-1 protease inhibitor. LEX032 has the ability to inhibit both neutrophil elastase and cathepsin G, two major neutral serine proteases in neutrophils, as well as superoxide generation. LEX032 (25 or 50 mg/kg) administered i.v. 1 min before reperfusion significantly attenuated myocardial necrotic injury evaluated by cardiac creatine kinase loss compared to MI/R rats receiving only vehicle (P LEX032 as compared with rats receiving vehicle (P LEX032 also moderately attenuated leukotriene B4-stimulated PMN adherence to rat superior mesenteric artery endothelium and markedly diminished superoxide radical release from LTB4-stimulated PMN in vitro. In a glycogen-induced rat peritonitis model, LEX032 (50 mg/kg) significantly attenuated PMN transmigration into the peritoneal cavity in vivo. In conclusion, the recombinant serine protease inhibitor, LEX032, appears to be an effective agent for attenuating MI/R injury by inhibiting neutrophil-accumulation into the ischemic-reperfused myocardium and by inactivating cytotoxic metabolites (proteases and superoxide radical) released from neutrophils.

  2. 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...

  3. Inhibition of Hsp27 Radiosensitizes Head-and-Neck Cancer by Modulating Deoxyribonucleic Acid Repair

    Energy Technology Data Exchange (ETDEWEB)

    Guttmann, David M.; Hart, Lori [Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Du, Kevin [Department of Radiation Oncology, New York University School of Medicine, New York, New York (United States); Seletsky, Andrew [Department of Biology, Drexel University, Philadelphia, Pennsylvania (United States); Koumenis, Constantinos, E-mail: koumenis@xrt.upenn.edu [Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States)

    2013-09-01

    Purpose: To present a novel method of tumor radiosensitization through Hsp27 knockdown using locked nucleic acid (LNA) and to investigate the role of Hsp27 in DNA double strand break (DSB) repair. Methods and Materials: Clonogenic survival assays, immunoblotting, the proximity ligation assay, and γH2AX foci analysis were conducted in SQ20B and FaDu human head-and-neck cancer cell lines treated with Hsp27 LNA and Hsp27 short hairpin RNA (shRNA). Additionally, nude mice with FaDu flank tumors were treated with fractionated radiation therapy after pretreatment with Hsp27 LNA and monitored for tumor growth. Results: Hsp27 LNA and Hsp27 shRNA radiosensitized head-and-neck cancer cell lines in an Hsp27-dependent manner. Ataxia-Telangectasia Mutated-mediated DNA repair signaling was impaired in irradiated cells with Hsp27 knockdown. ATM kinase inhibition abrogated the radiosensitizing effect of Hsp27. Furthermore, Hsp27 LNA and shRNA both attenuated DNA repair kinetics after radiation, and Hsp27 was found to colocalize with ATM in both untreated and irradiated cells. Last, combined radiation and Hsp27 LNA treatment in tumor xenografts in nude mice suppressed tumor growth compared with either treatment alone. Conclusions: These results support a radiosensitizing property of Hsp27 LNA in vitro and in vivo, implicate Hsp27 in double strand break repair, and suggest that Hsp27 LNA might eventually serve as an effective clinical agent in the radiotherapy of head-and-neck cancer.

  4. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    Science.gov (United States)

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  5. Mechanisms of Oxidative Damage in Multiple Sclerosis and Neurodegenerative Diseases: Therapeutic Modulation via Fumaric Acid Esters

    Directory of Open Access Journals (Sweden)

    Ralf Gold

    2012-09-01

    Full Text Available Oxidative stress plays a crucial role in many neurodegenerative conditions such as Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s as well as Huntington’s disease. Inflammation and oxidative stress are also thought to promote tissue damage in multiple sclerosis (MS. Recent data point at an important role of anti-oxidative pathways for tissue protection in chronic-progressive MS, particularly involving the transcription factor nuclear factor (erythroid-derived 2-related factor 2 (Nrf2. Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for MS treatment. Here, fumaric acid esters (FAE are a new, orally available treatment option which had already been tested in phase II/III MS trials demonstrating beneficial effects on relapse rates and magnetic resonance imaging markers. In vitro, application of dimethylfumarate (DMF leads to stabilization of Nrf2, activation of Nrf2-dependent transcriptional activity and abundant synthesis of detoxifying proteins. Furthermore, application of FAE involves direct modification of the inhibitor of Nrf2, Kelch-like ECH-associated protein 1. On cellular levels, the application of FAE enhances neuronal survival and protects astrocytes against oxidative stress. Increased levels of Nrf2 are detected in the central nervous system of DMF treated mice suffering from experimental autoimmune encephalomyelitis (EAE, an animal model of MS. In EAE, DMF ameliorates the disease course and improves preservation of myelin, axons and neurons. Finally, Nrf2 is also up-regulated in the spinal cord of autopsy specimens from untreated patients with MS, probably as part of a naturally occurring anti-oxidative response. In summary, oxidative stress and anti-oxidative pathways are important players in MS pathophysiology and constitute a promising target for future MS therapies like FAE.

  6. 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.

  7. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    Science.gov (United States)

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. 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.

  9. A Salmonella Regulator Modulates Intestinal Colonization and Use of Phosphonoacetic Acid

    Science.gov (United States)

    Elfenbein, Johanna R.; Knodler, Leigh A.; Schaeffer, Allison R.; Faber, Franziska; Bäumler, Andreas J.; Andrews-Polymenis, Helene L.

    2017-01-01

    Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of phosphonates in the marine biosphere is well characterized but the role of these molecules in the intestine is poorly understood. Salmonella enterica uses its virulence factors to influence the host immune response to compete with the host and normal microflora for nutrients. Salmonella cannot produce phosphonates but encodes the enzymes to use them suggesting that it is exposed to phosphonates during its life cycle. The role of phosphonates during enteric salmonellosis is unexplored. We have previously shown that STM3602, encoding a putative regulator of phosphonate metabolism, is needed for colonization in calves. Here, we report that the necessity of STM3602 in colonization of the murine intestine results from multiple factors. STM3602 is needed for full activation of the type-3 secretion system-1 and for optimal invasion of epithelial cells. The ΔSTM3602 mutant grows poorly in phosphonoacetic acid (PA) as the sole phosphorus source, but can use 2-aminoethylphosphonate. PhnA, an enzyme required for PA breakdown, is not controlled by STM3602 suggesting an additional mechanism for utilization of PA in S. Typhimurium. Finally, the requirement of STM3602 for intestinal colonization differs depending on the composition of the microflora. Our data suggest that STM3602 has multiple regulatory targets that are necessary for survival within the microbial community in the intestine. Determination of the members of the STM3602 regulon may illuminate new pathways needed for colonization of the host. PMID:28361036

  10. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    Science.gov (United States)

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  11. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  12. Stomatin-domain protein interactions with acid-sensing ion channels modulate nociceptor mechanosensitivity

    Science.gov (United States)

    Moshourab, Rabih A; Wetzel, Christiane; Martinez-Salgado, Carlos; Lewin, Gary R

    2013-01-01

    Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:stomatin−/− mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or stomatin−/− mice. Examination of stomatin−/−:stoml3−/− mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function. PMID:23959680

  13. A cysteine protease isolated from the latex of Ficus microcarpa: purification and biochemical characterization.

    Science.gov (United States)

    Mnif, Ibtissem Hamza; Siala, Rayda; Nasri, Rim; Mhamdi, Samiha; Nasri, Moncef; Kamoun, Alya Sellami

    2015-02-01

    A plant protease named microcarpain was purified from the latex of Ficus microcarpa by acetonic (20-40 % saturation) precipitation, Sephadex G-75 filtration, and Mono Q-Sefinose FF chromatography. The protease was purified with a yield of 9.25 % and a purification factor of 8. The molecular weight of the microcarpain was estimated to be 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed maximum activity at pH 8.0 and at a temperature of 70 °C. Proteolytic activity was strongly inhibited by dithio-bis-nitrobenzoic acid (DTNB), Hg(2+), and Cu(2+). The N-terminal amino acid sequence of the purified microcarpain "VPETVDWRSKGAV" showed high homology with a protease from Arabidopsis thaliana. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine peptidases family.

  14. 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.

  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. Modulation of fibroblast growth factor 19 expression by bile acids, meal replacement and energy drinks, milk, and coffee.

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    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.

  17. Salicylic acid modulates arsenic toxicity by reducing its root to shoot translocation in rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Amit Pal Singh

    2015-05-01

    Full Text Available Arsenic (As is posing serious health concerns in South East Asia where rice, an efficient accumulator of As, is prominent crop. Salicylic acid (SA is an important signalling molecule and plays a crucial role in resistance against biotic and abiotic stress in plants. In present study, ameliorative effect of SA against arsenate (AsV toxicity has been investigated in rice (Oryza sativa L.. Arsenate stress hampered the plant growth in terms of root, shoots length and biomass as well as it enhanced the level of H2O2 and MDA in dose dependent manner in shoot. Exogenous application of SA, reverted the growth and oxidative stress caused byAsV and significantly decreased As translocation to the shoots. Level of As in shoot was positively correlated with the expression of OsLsi2, efflux transporter responsible for root to shoot translocation of arsenic in the form of arsenite (AsIII. Salicylic acid also overcame AsV induced oxidative stress and modulated the activities of antioxidant enzymes in a differential manner in shoots. Arsenic treatment hampered the translocation of Fe in the shoot which was compensated by the SA treatment. The level of Fe in root and shoot was positively correlated with the transcript level of transporters responsible for the accumulation of Fe,OsNRAMP5 and OsFRDL1, in the root and shoot respectively. Co-application of SA was more effective than pre-treatment for reducing As accumulation as well as imposed toxicity.

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

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    Byoung-Kuk Na

    Full Text Available BACKGROUND: 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. FINDINGS: 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. CONCLUSION: VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates

  19. Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased Influenza A virus cleavage and replication

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    Kesic Matthew J

    2012-09-01

    Full Text Available Abstract Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.

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

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    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

  1. 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

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

    Science.gov (United States)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    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. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  3. Oral administration of Bifidobacterim bifidum for modulating microflora, acid and bile resistance, and physiological indices in mice.

    Science.gov (United States)

    Wang, Bao-Gui; Xu, Hai-Bo; Wei, Hua; Zeng, Zhe-Ling; Xu, Feng

    2015-02-01

    Bifidobacteria are generally acknowledged as major gut microflora used as probiotics, which promote human health. In this study, the effects of the administration of Bifidobacterim bifidum on modulating gastrointestinal (GI) tract microflora, acid and bile resistance, and physiological indices in BALB/c mice were investigated. Results showed that B. bifidum can significantly improve the ecosystem of the GI tract by increasing the amount of probiotics and reducing the populations of pathogenic bacteria, as measured by plate count and real-time PCR. After exposure to simulated GI tract conditions, the growth of gut microflora in the B. bifidum group was higher than that in the control group when incubated for 12 h in MRS or nutrient broth adjusted to pH 2.0 or 3.0 or in the presence of a concentration of bile salt (0.45% m/v). The blood biochemical index was examined, and the physiological effect of the cell-free extract of gut microflora was evaluated by measuring the activity of various enzymes, including α-glucosidases, esterase, and lactate dehydrogenase. This study suggested that a B. bifidum strain can stabilize blood sugar, lower cholesterol levels in serum, and improve metabolic activity. Moreover, B. bifidum was a promising enhancer of microbial diversity in mouse intestine and played a vital role in human physiological processes, which can benefit the health of a host.

  4. Postbiotic Modulation of Retinoic Acid Imprinted Mucosal-like Dendritic Cells by Probiotic Lactobacillus reuteri 17938 In vitro

    Directory of Open Access Journals (Sweden)

    Yeneneh eHaileselassie

    2016-03-01

    Full Text Available Lactobacilli are widely used as probiotics with beneficial effects on infection-associated diarrhea, but also used in clinical trials of e.g. necrotizing enterocolitis and inflammatory bowel diseases. The possibility of using probiotic metabolic products, so called postbiotics, is desirable as it could prevent possible side effects of live bacteria in individuals with a disturbed gut epithelial barrier. Here we studied how Lactobacillus reuteri DSM 17938 cell free supernatant (L. reuteri-CFS influenced retinoic acid (RA-driven mucosal-like dendritic cells (DC and their subsequent effect on T regulatory cells (Treg in vitro. RA clearly imprinted a mucosal-like DC phenotype with higher IL10 production, increased CD103 and CD1d expression and a down-regulated mRNA expression of several inflammatory-associated genes (NFκB1, RELB and TNF. Treatment with L. reuteri-CFS further influenced the tolerogenic phenotype of RA-DC by down-regulating most genes involved in antigen uptake, antigen presentation and signal transduction as well as several chemokine receptors, while up regulating IL10 production. L. reuteri-CFS also augmented CCR7 expression on RA-DC. In co-cultures, RA-DC increased IL10 and FOXP3 expression in Treg, but pre-treatment with L. reuteri-CFS did not further influence the Treg phenotype. In conclusion, L. reuteri-CFS modulates the phenotype and function of mucosal-like DC, implicating its potential application as postbiotic.

  5. Postbiotic Modulation of Retinoic Acid Imprinted Mucosal-like Dendritic Cells by Probiotic Lactobacillus reuteri 17938 In Vitro.

    Science.gov (United States)

    Haileselassie, Yeneneh; Navis, Marit; Vu, Nam; Qazi, Khaleda Rahman; Rethi, Bence; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are widely used as probiotics with beneficial effects on infection-associated diarrhea, but also used in clinical trials of e.g., necrotizing enterocolitis and inflammatory bowel diseases. The possibility of using probiotic metabolic products, so-called postbiotics, is desirable as it could prevent possible side effects of live bacteria in individuals with a disturbed gut epithelial barrier. Here, we studied how Lactobacillus reuteri DSM 17938 cell-free supernatant (L. reuteri-CFS) influenced retinoic acid (RA)-driven mucosal-like dendritic cells (DC) and their subsequent effect on T regulatory cells (Treg) in vitro. RA clearly imprinted a mucosal-like DC phenotype with higher IL10 production, increased CD103 and CD1d expression, and a downregulated mRNA expression of several inflammatory-associated genes (NFκB1, RELB, and TNF). Treatment with L. reuteri-CFS further influenced the tolerogenic phenotype of RA-DC by downregulating most genes involved in antigen uptake, antigen presentation, and signal transduction as well as several chemokine receptors, while upregulating IL10 production. L. reuteri-CFS also augmented CCR7 expression on RA-DC. In cocultures, RA-DC increased IL10 and FOXP3 expression in Treg, but pre-treatment with L. reuteri-CFS did not further influence the Treg phenotype. In conclusion, L. reuteri-CFS modulates the phenotype and function of mucosal-like DC, implicating its potential application as postbiotic.

  6. 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.

  7. Structural basis for the immunomodulatory function of cysteine protease inhibitor from human roundworm Ascaris lumbricoides.

    Science.gov (United States)

    Mei, Guoqiang; Dong, Jianmei; Li, Zhaotao; Liu, Sanling; Liu, Yunfeng; Sun, Mingze; Liu, Guiyun; Su, Zhong; Liu, Jinsong

    2014-01-01

    Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.

  8. Differential modulation of alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors expressed in Xenopus oocytes by flufenamic acid and niflumic acid.

    Science.gov (United States)

    Zwart, R; Oortgiesen, M; Vijverberg, H P

    1995-03-01

    Effects of flufenamic acid (FFA) and niflumic acid (NFA), which are often used to block Ca(2+)-activated Cl- current, have been investigated in voltage-clamped Xenopus oocytes expressing alpha 3 beta 2 and alpha 3 beta 4 nicotinic ACh receptors (nAChRs). NFA and FFA inhibit alpha 3 beta 2 nAChR-mediated inward currents and potentiate alpha 3 beta 4 nAChR-mediated inward currents in normal, Cl(-)-free and Ca(2+)-free solutions to a similar extent. The concentration-dependence of the inhibition of alpha 3 beta 2 nAChR-mediated ion current yields IC50 values of 90 microM for FFA and of 260 microM for NFA. The potentiation of alpha 3 beta 4 nAChR-mediated ion current by NFA yields an EC50 value of 30 microM, whereas the effect of FFA does not saturate for concentrations of up to 1 mM. At 100 microM, FFA reduces the maximum of the concentration-effect curve of ACh for alpha 3 beta 2 nAChRs, but leaves the EC50 of ACh unaffected. The same concentration of FFA potentiates alpha 3 beta 4 nAChR-mediated ion currents for all ACh concentrations and causes a small shift of the concentration-effect curve of ACh to lower agonist concentrations. The potentiation, like the inhibition, is most likely due to a noncompetitive effect of FFA. Increasing ACh-induced inward current either by raising the agonist concentration from 10 microM to 200 microM or by coapplication of 10 microM ACh and 200 microM FFA causes a similar enhancement of block of the alpha 3 beta 4 nAChR-mediated ion current by Mg2+. This suggests that the effects of FFA and of an increased agonist concentration result in a similar functional modification of the alpha 3 beta 4 nAChR-operated ion channel. It is concluded that alpha 3 beta 4 and alpha 3 beta 2 nAChRs are oppositely modulated by FFA and NFA through a direct beta-subunit-dependent effect.

  9. 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.

  10. The role of proteases in the differentiation of Acanthamoeba castellanii.

    Science.gov (United States)

    Dudley, Ricky; Alsam, Selwa; Khan, Naveed Ahmed

    2008-09-01

    Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

  11. Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction

    Directory of Open Access Journals (Sweden)

    Jindřich Chmelař

    2017-05-01

    Full Text Available The publication of the first tick sialome (salivary gland transcriptome heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

  12. 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...... for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity...... and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained...

  13. The effect of an exogenous protease on the fermentation and nutritive value of high-moisture corn.

    Science.gov (United States)

    Kung, L; Windle, M C; Walker, N

    2014-03-01

    The objective of this study was to determine if treating high-moisture corn at harvest with an exogenous protease could accelerate the increase in in vitro ruminal starch degradation that is normally found with advancing times of ensiling. Ground high-moisture corn (HMC; 73% dry matter) was untreated or treated with an exogenous protease to achieve a final concentration of 2,000 mg of protease/kg of fresh corn. Corn was ensiled in laboratory-scale bags (approximately 500 g) that were evacuated of air, heat-sealed, and stored at 22 to 23°C for 70 and 140 d. Samples of freshly treated corn samples were collected to represent d 0 samples. Treatment of HMC with protease did not affect the gross populations of lactic acid bacteria or yeasts throughout the ensiling period. Treatment of HMC with protease resulted in higher concentrations of lactic acid and ethanol after 70 but not 140 d of ensiling. Concentrations of crude protein, water-soluble carbohydrates, and starch were unaffected by treatment with protease within each sampling day. After 70 or 140 d of ensiling, HMC that was treated with protease had higher concentrations of soluble protein (as a % of crude protein) and NH3-N, and had lower concentrations of prolamin protein, compared with untreated corn. In vitro rumen degradability (7-h incubation) of starch was greater in protease-treated versus untreated corn at all sampling days but the difference was more pronounced after 70 and 140 d compared with d 0. Concentrations of soluble protein and NH3-N were positively correlated with in vitro starch degradation. Conversely, the concentrations of prolamin protein in HMC were negatively correlated with in vitro starch degradation. Treating HMC with an exogenous protease could be a method to obtain greater potential for ruminal starch fermentation after a relatively short period of ensiling. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Blunting the knife: development of vaccines targeting digestive proteases of blood-feeding helminth parasites.

    Science.gov (United States)

    Pearson, Mark S; Ranjit, Najju; Loukas, Alex

    2010-08-01

    Proteases are pivotal to parasitism, mediating biological processes crucial to worm survival including larval migration through tissue, immune evasion/modulation and nutrient acquisition by the adult parasite. In haematophagous parasites, many of these proteolytic enzymes are secreted from the intestine (nematodes) or gastrodermis (trematodes) where they act to degrade host haemoglobin and serum proteins as part of the feeding process. These proteases are exposed to components of the immune system of the host when the worms ingest blood, and therefore present targets for the development of anti-helminth vaccines. The protective effects of current vaccine antigens against nematodes that infect humans (hookworm) and livestock (barber's pole worm) are based on haemoglobin-degrading intestinal proteases and act largely as a result of the neutralisation of these proteases by antibodies that are ingested with the blood-meal. In this review, we survey the current status of helminth proteases that show promise as vaccines and describe their vital contribution to a parasitic existence.

  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.

    2013-01-01

    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. PMID:24688526

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

    Directory of Open Access Journals (Sweden)

    Anderson F. Santos

    2013-12-01

    Full Text Available 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.

  17. Extreme entropy-enthalpy compensation in a drug-resistant variant of HIV-1 protease.

    Science.gov (United States)

    King, Nancy M; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M; Nalam, Madhavi N L; Nalivaika, Ellen A; Özen, Ayşegül; Haliloğlu, Türkan; Yilmaz, Neşe Kurt; Schiffer, Celia A

    2012-09-21

    The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.

  18. Intestinal fatty acid infusion modulates food preference as well as calorie intake via the vagal nerve and midbrain-hypothalamic neural pathways in rats.

    Science.gov (United States)

    Ogawa, Nobuya; Ito, Makoto; Yamaguchi, Hideki; Shiuchi, Tetsuya; Okamoto, Shiki; Wakitani, Korekiyo; Minokoshi, Yasuhiko; Nakazato, Masamitsu

    2012-09-01

    The intestine plays important roles in the regulation of feeding behavior by sensing macronutrients. Intestinal fatty acids strongly suppress food intake, but little is known about whether intestinal fatty acids affect food preference. We investigated the effects of jejunal fatty acids infusion on food preference by conducting two-diet choice experiments in rats fed a high-fat diet (HFD) and a high-carbohydrate diet (HCD). Jejunal linoleic acid (18:2) infusion reduced HFD intake dose-dependently, while HCD intake increased with the middle dose of the infusion we examined (100 μL/h) and reduced to the control level with the higher doses (150 and 200 μL/h). α-Linolenic acid (18:3), but not caprylic acid (8:0), altered the food preference and total calorie intake in the same manner as linoleic acid. Linoleic acid infusion dose-dependently increased plasma glucagon-like peptide-1, peptide YY and cholecystokinin levels, but not ghrelin levels. Subdiaphragmatic vagotomy or midbrain transection prevented the change in food preference and total calorie intake by linoleic acid infusion. Jejunal linoleic acid infusion increased norepinephrine turnover in the paraventricular hypothalamic nucleus, while intracerebroventricular injection of idazoxan, an α2-adrenergic receptor (AR) antagonist, suppressed the increased HCD intake, but did not affect the decreased HFD intake. These findings indicated that intestinal long-chain fatty acids modulated food preference as well as total calorie intake via the vagal nerve and midbrain-hypothalamic neural pathways. The effects of the α2-AR antagonist in the brain suggested that the brain distinctly controlled HCD and HFD intake in response to jejunal linoleic acid infusion. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Evidence of protease in the saliva of the butterfly Heliconius melpomene (L.) (Nymphalidae, Lepidoptera).

    Science.gov (United States)

    Eberhard, S H; Hrassnigg, N; Crailsheim, K; Krenn, H W

    2007-02-01

    Butterflies of the genus Heliconius are well known for their peculiar habits of utilizing pollen as a source of amino acids. Saliva plays a major role in the process of extracting amino acids and proteins from the pollen grains. In this investigation, we obtained samples of saliva from adult Heliconius melpomene by placing pumpkin pollen or fine glass-beads on the proboscis, which stimulates the butterflies to release saliva. Proteolytic activity was determined in the saliva by an insoluble protein-dye that turns blue when cleaved by proteases. Its extinction value was measured with a spectrophotometer at 595 nm. Both the saliva sampled with pollen and the saliva obtained from inert glass-beads exhibit proteolytic activity demonstrating that the saliva contains proteases. The proteolytic activity of the pollen/saliva samples was higher than that of the glass-bead/saliva samples, which we attribute to the stimulating effects of pollen, such as taste, smell, and texture, and not to proteases which might have been liberated from the pollen. This is indicated by the fact that pollen samples without saliva showed only a negligible indication for proteolytic activity. In general, females exhibit higher proteolytic activities than males, presumably due to their greater amino acid investment in reproduction. We present here first evidence for the existence of proteases in the saliva of a butterfly species and suggest that these enzymes are crucial for the use of amino acids and proteins from pollen in Heliconius butterflies.

  20. Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Groot, R. de; Osterhaus, A.D.; Reedijk, M.L.; Verschuren, E.J.; Gruters, R.A.; Luider, T.M.

    2008-01-01

    In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and p

  1. MALT1 protease: equilibrating immunity versus tolerance

    OpenAIRE

    Bertossi, Arianna; Krappmann, Daniel

    2014-01-01

    MALT1 paracaspase links signaling cascades emanating from adaptive or innate immune receptors to the canonical NF-κB pathway. Now, Jaworski et al (2014) investigate the physiological role of MALT1 protease activity in mice. Besides the expected requirement of MALT1 activity for immune activation, the study unveils a novel function for MALT1 activity for the development of peripheral tolerance. Thus, MALT1 protease can act immunogenic or tolerogenic, and this interplay will be highly relevant ...

  2. 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

    2014-07-01

    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 Ca(2+)-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 Ca(2+) 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 Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-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.

  3. The Silica-Water Interface: How the Silanols Determine the Surface Acidity and Modulate the Water Properties.

    Science.gov (United States)

    Sulpizi, Marialore; Gaigeot, Marie-Pierre; Sprik, Michiel

    2012-03-13

    Silica is the most abundant metal oxide and the main component of the Earth's crust. Its behavior in contact with water plays a critical role in a variety of geochemical and environmental processes. Despite its key role, the details of the aqueous silica interface at the microscopic molecular level are still elusive. Here we provide such a detailed understanding of the molecular behavior of the silica-water interface, using density functional theory based molecular dynamics (DFTMD) simulations, where a consistent treatment of the electronic structure of solvent and surface is provided. We have calculated the acidity of the silanol groups at the interface directly from the DFTMD simulations, without any fitting of parameters to the experimental data. We find two types of silanol groups at the surface of quartz: out-of-plane silanols with a strong acidic character (pKa = 5.6), which consequently results in the formation of strong and short hydrogen bonds with water molecules at the interface, and in-plane silanols with a pKa of 8.5, forming weak hydrogen bonds with the interfacial water molecules. Our estimate of the quartz point of zero charge (1.0) is found in good agreement with the experimental value of 1.9. We have also shown how the silanols orientation and their hydrogen bond properties are responsible for an amphoteric behavior of the surface. A detailed analysis has identified two species of adsorbed water molecules at the solid-liquid interface, which using the language of vibrational spectroscopy can be identified as "liquid-like" and "ice-like" water or, in other words, water molecules forming respectively weak and strong H-bonds with the oxide surface. These two populations of water are in turn responsible for two distinct peaks in the infrared spectrum of interfacial water and thus provide a molecular explanation of the experimental sum frequency generation spectrum recorded in the literature. In the specific case of quartz, we show that the liquid

  4. Cancer procoagulant and tissue factor are differently modulated by all-trans-retinoic acid in acute promyelocytic leukemia cells.

    Science.gov (United States)

    Falanga, A; Consonni, R; Marchetti, M; Locatelli, G; Garattini, E; Passerini, C G; Gordon, S G; Barbui, T

    1998-07-01

    All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4. 306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] alpha), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARbeta) and CD437 (selective for RARgamma), both lacking this capability. Cells were treated with either ATRA or the analogues (10(-6) to 10(-8) mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARalpha agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA

  5. Modulation of growth and angiogenic potential of oral squamous carcinoma cells in vitro using salvianolic acid B

    Science.gov (United States)

    2011-01-01

    Background Our previous studies showed that Salvianolic acid B (Sal B) inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters and such anti-cancer effects might be related to the inhibition of angiogenesis. This study was aimed to further investigate the anti-proliferative effect of Sal B on the most common type of oral cancer, oral squamous cell carcinoma (OSCC) and the possible mechanisms of action with respect to angiogenesis inhibition. Methods Two well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC4, and premalignant leukoplakia cells were treated with different concentrations of Sal B. Cytotoxicity was assessed by MTT assay. cDNA microarray was utilized to evaluate the expression of 96 genes known to be involved in modulating the biological processes of angiogenesis. Real-time reverse transcription-polymerase chain reaction analysis was conducted to confirm the cDNA microarray data. Results Sal B induced growth inhibition in OSCC cell lines but had limited effects on premalignant cells. A total of 17 genes showed a greater than 3-fold change when comparing Sal B treated OSCC cells to the control. Among these genes, HIF-1α, TNFα and MMP9 are specifically inhibited, expression of THBS2 was up-regulated. Conclusions Sal B has inhibitory effect on OSCC cell growth. The antitumor effect can be attributed to anti-angiogenic potential induced by a decreased expression of some key regulator genes of angiogenesis. Sal B may be a promising modality for treating oral squamous cell carcinoma. PMID:21726465

  6. Modulation of growth and angiogenic potential of oral squamous carcinoma cells in vitro using salvianolic acid B

    Directory of Open Access Journals (Sweden)

    Zhu Qin Y

    2011-07-01

    Full Text Available Abstract Background Our previous studies showed that Salvianolic acid B (Sal B inhibited 7,12-dimethylbenz[a]anthracene (DMBA-induced oral carcinogenesis in hamsters and such anti-cancer effects might be related to the inhibition of angiogenesis. This study was aimed to further investigate the anti-proliferative effect of Sal B on the most common type of oral cancer, oral squamous cell carcinoma (OSCC and the possible mechanisms of action with respect to angiogenesis inhibition. Methods Two well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC4, and premalignant leukoplakia cells were treated with different concentrations of Sal B. Cytotoxicity was assessed by MTT assay. cDNA microarray was utilized to evaluate the expression of 96 genes known to be involved in modulating the biological processes of angiogenesis. Real-time reverse transcription-polymerase chain reaction analysis was conducted to confirm the cDNA microarray data. Results Sal B induced growth inhibition in OSCC cell lines but had limited effects on premalignant cells. A total of 17 genes showed a greater than 3-fold change when comparing Sal B treated OSCC cells to the control. Among these genes, HIF-1α, TNFα and MMP9 are specifically inhibited, expression of THBS2 was up-regulated. Conclusions Sal B has inhibitory effect on OSCC cell growth. The antitumor effect can be attributed to anti-angiogenic potential induced by a decreased expression of some key regulator genes of angiogenesis. Sal B may be a promising modality for treating oral squamous cell carcinoma.

  7. Salicylic acid is a modulator of catalase isozymes in chickpea plants infected with Fusarium oxysporum f. sp. ciceri.

    Science.gov (United States)

    Gayatridevi, S; Jayalakshmi, S K; Sreeramulu, K

    2012-03-01

    The relationship between salicylic acid level catalases isoforms chickpea cv. ICCV-10 infected with Fusarium oxysporum f. sp. ciceri was investigated. Pathogen-treated chickpea plants showed high levels of SA compared with the control. Two isoforms of catalases in shoot extract (CAT-IS and CAT-IIS) and single isoform in root extract (CAT-R) were detected in chickpea. CAT-IS and CAT-R activities were inhibited in respective extracts treated with pathogen whereas, CAT-IIS activity was not inhibited. These isoforms were purified and their kinetic properties studied in the presence or absence of SA. The molecular mass determined by SDS-PAGE of CAT-IS, CAT-IIS and CAT-R was found to be 97, 40 and 66 kDa respectively. Kinetic studies indicated that Km and V(max) of CAT-IS were 0.2 mM and 300 U/mg, 0.53 mM and 180 U/mg for CAT-IIS and 0.25 mM and 280 U/mg for CAT-R, respectively. CAT-IS and CAT-R were found to be more sensitive to SA and 50% of their activities were inhibited at 6 and 4 μM respectively, whereas CAT-IIS was insensitive to SA up to 100 μM. Quenching of the intrinsic tryptophan fluorescence of purified catalases were used to quantitate SA binding; the estimated K(d) value for CAT-IS, CAT-IIS and CAT-R found to be 2.3 μM, 3.1 mM and 2.8 μM respectively. SA is a modulator of catalase isozymes activity, supports its role in establishment of SAR in chickpea plants infected with the pathogen.

  8. Additive effect of polymorphisms in the IL-6, LTA, and TNF-{alpha} genes and plasma fatty acid level modulate risk for the metabolic syndrome and its components

    OpenAIRE

    2010-01-01

    Context: Cytokine polymorphisms and dietary fat composition may influence the risk of the metabolic syndrome (MetS). Objective: The objective of the study was to determine the relationship between lymphotoxin-α (LTA), TNF-α, and IL-6 gene polymorphisms with MetS risk and investigate whether plasma fatty acid composition, a biomarker of dietary fat intake, modulated these associations. Design: Polymorphisms (LTA rs915654, TNF-α rs1800629, IL-6 rs1800797), biochemical measurements, and plasma f...

  9. 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.

  10. 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.

  11. Biased signaling by peptide agonists of protease activated receptor 2.

    Science.gov (United States)

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  12. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

    Science.gov (United States)

    Neveu, Julie; Regeard, Christophe; DuBow, Michael S

    2011-08-01

    The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

  13. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    Science.gov (United States)

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, act