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Sample records for acid fungal protease

  1. A biotechnology perspective of fungal proteases

    PubMed Central

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira, Edivaldo Ximenes; Pessoa, Adalberto; Magalhães, Pérola Oliveira

    2015-01-01

    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. PMID:26273247

  2. A biotechnology perspective of fungal proteases.

    PubMed

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira Filho, Edivaldo Ximenes; Pessoa Junior, Adalberto; Magalhães, Pérola Oliveira

    2015-06-01

    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. PMID:26273247

  3. Secreted fungal aspartic proteases: A review.

    PubMed

    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. PMID:27137097

  4. Secreted fungal aspartic proteases: A review.

    PubMed

    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.

  5. Production of bioactive peptide hydrolysates from deer, sheep and pig plasma using plant and fungal protease preparations.

    PubMed

    Bah, Clara S F; Bekhit, Alaa El-Din A; Carne, Alan; McConnell, Michelle A

    2015-06-01

    Plasma separated from deer, sheep and pig blood, obtained from abattoirs, was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24h of hydrolysis, were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, while the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher DPPH radical-scavenging, oxygen radical-scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates. The results indicated that proteolytic hydrolysis of animal blood plasmas, using fungal protease preparations in particular, produces hydrolysates with high antioxidant properties.

  6. Production of bioactive peptide hydrolysates from deer, sheep and pig plasma using plant and fungal protease preparations.

    PubMed

    Bah, Clara S F; Bekhit, Alaa El-Din A; Carne, Alan; McConnell, Michelle A

    2015-06-01

    Plasma separated from deer, sheep and pig blood, obtained from abattoirs, was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24h of hydrolysis, were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, while the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher DPPH radical-scavenging, oxygen radical-scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates. The results indicated that proteolytic hydrolysis of animal blood plasmas, using fungal protease preparations in particular, produces hydrolysates with high antioxidant properties. PMID:25624206

  7. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    PubMed Central

    Nirmal, Nilesh P.; Laxman, R. Seeta

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory. PMID:25105022

  8. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.

    PubMed

    Mueller, André N; Ziemann, Sebastian; Treitschke, Steffi; Aßmann, Daniela; Doehlemann, Gunther

    2013-02-01

    The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi.

  9. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    PubMed

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity. PMID:27278806

  10. Generation of bioactive peptide hydrolysates from cattle plasma using plant and fungal proteases.

    PubMed

    Bah, Clara S F; Bekhit, Alaa El-Din A; McConnell, Michelle A; Carne, Alan

    2016-12-15

    Four protease preparations from plant and fungal sources (papain, bromelain, FP400 and FPII) were used to hydrolyse plasma which was separated from slaughterhouse cattle blood. The o-phthaldialdehyde assay was used to follow the release of TCA-soluble peptides over a 24h period. Hydrolysis profiles were displayed using SDS-PAGE. The in vitro antioxidant and antimicrobial activities of the hydrolysates were determined. The results showed that hydrolysates of cattle plasma generated with fungal protease FPII had higher antioxidant activities. Overall than hydrolysates generated with papain, bromelain and FP400. None of the hydrolysates demonstrated antimicrobial activity. The FPII peptide hydrolysate was fractionated using gel permeation chromatography, OFFGEL isoelectric focusing and RP-HPLC. The RP-HPLC fraction with highest antioxidant activity contained 15 novel peptide sequences. The use of protease FPII to hydrolyse cattle plasma resulted in a hydrolysate with high antioxidant properties and unique peptide sequences. PMID:27451160

  11. Generation of bioactive peptide hydrolysates from cattle plasma using plant and fungal proteases.

    PubMed

    Bah, Clara S F; Bekhit, Alaa El-Din A; McConnell, Michelle A; Carne, Alan

    2016-12-15

    Four protease preparations from plant and fungal sources (papain, bromelain, FP400 and FPII) were used to hydrolyse plasma which was separated from slaughterhouse cattle blood. The o-phthaldialdehyde assay was used to follow the release of TCA-soluble peptides over a 24h period. Hydrolysis profiles were displayed using SDS-PAGE. The in vitro antioxidant and antimicrobial activities of the hydrolysates were determined. The results showed that hydrolysates of cattle plasma generated with fungal protease FPII had higher antioxidant activities. Overall than hydrolysates generated with papain, bromelain and FP400. None of the hydrolysates demonstrated antimicrobial activity. The FPII peptide hydrolysate was fractionated using gel permeation chromatography, OFFGEL isoelectric focusing and RP-HPLC. The RP-HPLC fraction with highest antioxidant activity contained 15 novel peptide sequences. The use of protease FPII to hydrolyse cattle plasma resulted in a hydrolysate with high antioxidant properties and unique peptide sequences.

  12. Measuring Chitinase and Protease Activity in Cultures of Fungal Entomopathogens.

    PubMed

    Cheong, Peter; Glare, Travis R; Rostás, Michael; Haines, Stephen R

    2016-01-01

    Entomopathogenic fungi produce a variety of destructive enzymes and metabolites to overcome the unique defense mechanisms of insects. In a first step, fungal chitinases and proteinases need to break down the insect's cuticle. Both enzyme classes support the infection process by weakening the chitin barrier and by producing nutritional cleavage products for the fungus. In a second step, the pathogen can now mechanically penetrate the weakened cuticle and reach the insect's hemolymph where it starts proliferating. The critical enzymes chitinase and proteinase are also excreted into the supernatants of fungal cultures and can be used as indicators of virulence. Chromogenic assays adapted for 96-well microtiter plates that measure these enzymes provide a sensitive, fast, and easy screening method for evaluating the potential biocontrol activity of fungal isolates and may be considered as an alternative to laborious and time-consuming bioassays. Furthermore, monitoring fungal enzyme production in dependence of time, nutrient sources, or other factors can facilitate in establishing optimal growth and harvesting conditions for selected isolates with the aim of achieving maximum biocontrol activity. PMID:27565500

  13. Bumblebee venom serine protease increases fungal insecticidal virulence by inducing insect melanization.

    PubMed

    Kim, Jae Su; Choi, Jae Young; Lee, Joo Hyun; Park, Jong Bin; Fu, Zhenli; Liu, Qin; Tao, Xueying; Jin, Byung Rae; Skinner, Margaret; Parker, Bruce L; Je, Yeon Ho

    2013-01-01

    Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment. PMID:23626832

  14. Bumblebee Venom Serine Protease Increases Fungal Insecticidal Virulence by Inducing Insect Melanization

    PubMed Central

    Kim, Jae Su; Choi, Jae Young; Lee, Joo Hyun; Park, Jong Bin; Fu, Zhenli; Liu, Qin; Tao, Xueying; Jin, Byung Rae; Skinner, Margaret; Parker, Bruce L.; Je, Yeon Ho

    2013-01-01

    Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment. PMID:23626832

  15. Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies.

    PubMed

    Sabotič, Jerica; Ohm, Robin A; Künzler, Markus

    2016-01-01

    Fruiting bodies or sporocarps of dikaryotic (ascomycetous and basidiomycetous) fungi, commonly referred to as mushrooms, are often rich in entomotoxic and nematotoxic proteins that include lectins and protease inhibitors. These protein toxins are thought to act as effectors of an innate defense system of mushrooms against animal predators including fungivorous insects and nematodes. In this review, we summarize current knowledge about the structures, target molecules, and regulation of the biosynthesis of the best characterized representatives of these fungal defense proteins, including galectins, beta-trefoil-type lectins, actinoporin-type lectins, beta-propeller-type lectins and beta-trefoil-type chimerolectins, as well as mycospin and mycocypin families of protease inhibitors. We also present an overview of the phylogenetic distribution of these proteins among a selection of fungal genomes and draw some conclusions about their evolution and physiological function. Finally, we present an outlook for future research directions in this field and their potential applications in medicine and crop protection.

  16. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  17. Secretion of a fungal protease represents a complement evasion mechanism in cerebral aspergillosis.

    PubMed

    Rambach, Günter; Dum, David; Mohsenipour, Iradj; Hagleitner, Magdalena; Würzner, Reinhard; Lass-Flörl, Cornelia; Speth, Cornelia

    2010-04-01

    Complement represents a central immune weapon in the brain, but the high lethality of cerebral aspergillosis indicates a low efficacy of the antifungal complement attack. Studies with cerebrospinal fluid (CSF) samples derived from a patient with cerebral aspergillosis showed a degradation of complement proteins, implying that Aspergillus might produce proteases to evade their antimicrobial potency. Further investigations of this hypothesis showed that Aspergillus, when cultured in CSF to simulate growth conditions in the brain, secreted a protease that can cleave various complement proteins. Aspergillus fumigatus, the most frequent cause of cerebral aspergillosis, destroyed complement activity more efficiently than other Aspergillus species. The degradation of complement in CSF resulted in a drastic reduction of the capacity to opsonize fungal hyphae. Furthermore, the Aspergillus-derived protease could diminish the amount of complement receptor CR3, a surface molecule to mediate eradication of opsonized pathogens, on granulocytes and microglia. The lack of these prerequisites caused a significant decrease in phagocytosis of primary microglia. Additional studies implied that the complement-degrading activity shares many characteristics with the previously described alkaline protease Alp1. To improve the current therapy for cerebral aspergillosis, we tried to regain the antifungal effects of complement by repressing the secretion of this degrading activity. Supplementation of CSF with nitrogen sources rescued the complement proteins and abolished any cleavage. Glutamine or arginine are of special interest for this purpose since they represent endogenous substances in the CNS and might be included in a future supportive therapy to reduce the high lethality of cerebral aspergillosis. PMID:20303595

  18. Characterisation of novel fungal and bacterial protease preparations and evaluation of their ability to hydrolyse meat myofibrillar and connective tissue proteins.

    PubMed

    Ryder, Kate; Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2015-04-01

    The catalytic capability of four commercially available food-grade fungal and bacterial protease preparations (AFP, FPII, F60K and HT) was evaluated over a range of pH, temperature and substrate conditions using esterase and caseinolytic activity assays and time course hydrolysis over 120 and 60 min of myofibrillar and connective tissue proteins, respectively. The protease preparations displayed similar casein hydrolysis kinetics and were active in hydrolysing BODIPY-FL casein to varying extents at postmortem aging meat pH (5.0-6.0). All of the four proteases exhibited selective hydrolytic activity towards meat myofibrillar proteins including myosin and actin. Significant hydrolysis of two meat tenderisation protein markers troponin T and desmin by the four proteases was detected by western blot. The results obtained indicate that the new fungal protease preparations AFP and FPII, bacterial protease preparation HT and the new source of fungal protease preparation F60K have potential for use in meat tenderising applications.

  19. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

    PubMed

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides. PMID:26071808

  20. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

    PubMed

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

  1. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

  2. Fungal biotransformation of crude glycerol into malic acid.

    PubMed

    West, Thomas P

    2015-01-01

    Malic acid production from the biodiesel coproduct crude glycerol by Aspergillus niger ATCC 9142, ATCC 10577 and ATCC 12846 was observed to occur with the highest malic acid level acid being produced by A. niger ATCC 12846. Fungal biomass production from crude glycerol was similar, but ATCC 10577 produced the highest biomass. Fungal biotransformation of crude glycerol into the commercially valuable organic acid malic acid appeared feasible.

  3. Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

    PubMed

    Flausino, O A; Dufau, L; Regasini, L O; Petrônio, M S; Silva, D H S; Rose, T; Bolzani, V S; Reboud-Ravaux, M

    2012-01-01

    The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the β-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular β-sheets involved in protein-protein interactions.

  4. Production of bioactive peptide hydrolysates from deer, sheep, pig and cattle red blood cell fractions using plant and fungal protease preparations.

    PubMed

    Bah, Clara S F; Carne, Alan; McConnell, Michelle A; Mros, Sonya; Bekhit, Alaa El-Din A

    2016-07-01

    Protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources were used to hydrolyze the red blood cell fractions (RBCFs) separated from deer, sheep, pig, and cattle abattoir-sourced blood. After 1, 2, 4 and 24h of hydrolysis, the antioxidant and antibacterial activities of the peptide hydrolysates obtained were investigated. The increase in trichloroacetic acid-soluble peptides over the hydrolysis period was examined using the o-phthaldialdehyde (OPA) assay and the hydrolysis profiles were illustrated using SDS-PAGE. Papain generated RBCF hydrolysates exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) compared to those generated with bromelain, FP400 and FPII. At certain concentrations, 24h hydrolysates of RBCF using FP400 and FPII were able to inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results indicated that the use of proteases from plant or fungal sources can produce animal blood hydrolysates with antioxidant and antimicrobial activities. PMID:26920319

  5. Production of bioactive peptide hydrolysates from deer, sheep, pig and cattle red blood cell fractions using plant and fungal protease preparations.

    PubMed

    Bah, Clara S F; Carne, Alan; McConnell, Michelle A; Mros, Sonya; Bekhit, Alaa El-Din A

    2016-07-01

    Protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources were used to hydrolyze the red blood cell fractions (RBCFs) separated from deer, sheep, pig, and cattle abattoir-sourced blood. After 1, 2, 4 and 24h of hydrolysis, the antioxidant and antibacterial activities of the peptide hydrolysates obtained were investigated. The increase in trichloroacetic acid-soluble peptides over the hydrolysis period was examined using the o-phthaldialdehyde (OPA) assay and the hydrolysis profiles were illustrated using SDS-PAGE. Papain generated RBCF hydrolysates exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) compared to those generated with bromelain, FP400 and FPII. At certain concentrations, 24h hydrolysates of RBCF using FP400 and FPII were able to inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results indicated that the use of proteases from plant or fungal sources can produce animal blood hydrolysates with antioxidant and antimicrobial activities.

  6. Analysis of Free Amino Acid Pools in Fungal Mycelia

    PubMed Central

    Heathcote, J. G.; Davies, D. M.; Haworth, C.

    1972-01-01

    Free amino acid pools derived from three different types of fungal mycelia have been analyzed by the method of Heathcote and Haworth by using thinlayer chromatography. The preliminary extraction was carried out with boiling water and interfering proteins; peptides and salts were first removed by means of an ion-retardation resin. As far as determined, the results obtained represent the first quantitative analysis of fungal amino acid pools. PMID:4622829

  7. Vacuolar Serine Protease Is a Major Allergen of Fusarium proliferatum and an IgE-Cross Reactive Pan-Fungal Allergen

    PubMed Central

    Yeh, Chang-Ching; Tai, Hsiao-Yun; Chou, Hong; Wu, Keh-Gong

    2016-01-01

    Purpose Fusarium species are among prevalent airborne fungi and causative agents of human respiratory atopic disorders. We previously identified a 36.5-kDa F. proliferatum component recognized by IgE antibodies in 9 (53%) of the 17 F. proliferatum-sensitized atopic serum samples. The purpose of this study is to characterize the 36.5-kDa allergen of F. proliferatum. Methods Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning/expression and immunoblot inhibition studies. Results Based on the finding that the 36.5-kDa IgE-binding component reacted with the mouse monoclonal antibody FUM20 against fungal vacuolar serine protease allergens, the cDNA of F. proliferatum vacuolar serine protease (Fus p 9.0101) was subsequently cloned. Nine serum samples from respiratory atopic patients with IgE binding to the vacuolar serine protease allergen of Penicillium chrysogenum (Pen ch 18) also showed IgE-immunoblot reactivity to rFus p 9.0101. The purified rFus p 9.0101 can inhibit IgE and FUM20 binding to the 36.5-kDa component of F. proliferatum. Thus, a novel and important Fus p 9.0101 was identified. The rPen ch 18 can inhibit IgE binding to Fus p 9.0101. It indicates that IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 also exists. Furthermore, neither rFus p 9.0101 K88A nor rPen ch 18 K89A mutants inhibited IgE binding to rFus p 9.0101. Lys88 was considered a critical core amino acid in IgE binding to r Fus p 9.0101 and a residue responsible for IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 allergens. Conclusions Results obtained from this study indicate that vacuolar serine protease may be a major allergen of F. proliferatum and an important IgE cross-reactive pan-fungal allergen, and provide important bases for clinical diagnosis of fungal allergy. PMID:27334782

  8. Effects of L- and iso-ascorbic acid on meat protein hydrolyzing activity of four commercial plant and three microbial protease preparations.

    PubMed

    Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2014-04-15

    The present study investigated the effects of both l- and iso-ascorbic acid (AA) on the activity of four plant proteases (papain, bromelain, actinidin and zingibain) and three microbial proteases (Bacterial Protease G, Fungal 31,000 and Fungal 60,000) preparations using fluorescent-labelled casein, meat myofibrillar and connective tissue extracts to explore their effects on meat structure components upon treatment with individual proteases. While l-AA in the range 0.8-3.2mM inhibited the activity of papain, bromelain and zingibain, iso-AA acted as an inhibitor of papain but as an activator of zingibain and had no significant effect on bromelain. Both AA isoforms acted as an activator of the actinidin protease and the concentration of AA isoforms appeared to affect the level of activation of the protease. The effect of the two AA isoforms on collagen and myofibrillar protein hydrolyzing activity varied depending on the concentration of the two AA isoforms. The results indicate the ability to up and down regulate the activity of the investigated proteases by using an appropriate concentration of the AA isoform.

  9. Effects of L- and iso-ascorbic acid on meat protein hydrolyzing activity of four commercial plant and three microbial protease preparations.

    PubMed

    Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2014-04-15

    The present study investigated the effects of both l- and iso-ascorbic acid (AA) on the activity of four plant proteases (papain, bromelain, actinidin and zingibain) and three microbial proteases (Bacterial Protease G, Fungal 31,000 and Fungal 60,000) preparations using fluorescent-labelled casein, meat myofibrillar and connective tissue extracts to explore their effects on meat structure components upon treatment with individual proteases. While l-AA in the range 0.8-3.2mM inhibited the activity of papain, bromelain and zingibain, iso-AA acted as an inhibitor of papain but as an activator of zingibain and had no significant effect on bromelain. Both AA isoforms acted as an activator of the actinidin protease and the concentration of AA isoforms appeared to affect the level of activation of the protease. The effect of the two AA isoforms on collagen and myofibrillar protein hydrolyzing activity varied depending on the concentration of the two AA isoforms. The results indicate the ability to up and down regulate the activity of the investigated proteases by using an appropriate concentration of the AA isoform. PMID:24295669

  10. Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease.

    PubMed

    Usha, R; Singh, M

    1996-01-15

    Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca(2+)-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

  11. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Characterisation of novel fungal and bacterial protease preparations and evaluation of their ability to hydrolyse meat myofibrillar and connective tissue proteins.

    PubMed

    Ryder, Kate; Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2015-04-01

    The catalytic capability of four commercially available food-grade fungal and bacterial protease preparations (AFP, FPII, F60K and HT) was evaluated over a range of pH, temperature and substrate conditions using esterase and caseinolytic activity assays and time course hydrolysis over 120 and 60 min of myofibrillar and connective tissue proteins, respectively. The protease preparations displayed similar casein hydrolysis kinetics and were active in hydrolysing BODIPY-FL casein to varying extents at postmortem aging meat pH (5.0-6.0). All of the four proteases exhibited selective hydrolytic activity towards meat myofibrillar proteins including myosin and actin. Significant hydrolysis of two meat tenderisation protein markers troponin T and desmin by the four proteases was detected by western blot. The results obtained indicate that the new fungal protease preparations AFP and FPII, bacterial protease preparation HT and the new source of fungal protease preparation F60K have potential for use in meat tenderising applications. PMID:25442543

  14. Fungal arachidonic acid-rich oil: research, development and industrialization.

    PubMed

    Ji, Xiao-Jun; Ren, Lu-Jing; Nie, Zhi-Kui; Huang, He; Ouyang, Ping-Kai

    2014-09-01

    Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

  15. Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants[OPEN

    PubMed Central

    Li, Yurong; Kabbage, Mehdi; Liu, Wende; Dickman, Martin B.

    2016-01-01

    The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance. PMID:26739014

  16. An acidic amino acid-specific protease from germinating soybeans.

    PubMed

    Tan-Wilson, A L; Liu, X; Chen, R; Qi, X; Wilson, K A

    1996-05-01

    The degradation of the beta-conglycinin protein reserves in soybean seeds during germination and early growth begins with the proteolysis of its alpha and alpha' subunits by an enzyme called Protease C1. In the pathway, a number of proteolytic intermediates are produced and subsequently degraded. Determination of the N-terminal sequences of these intermediates provides insight regarding the requirements of the cleavage sites. The N-terminal sequence of three such proteolytic intermediates has been determined. The sequence has been located in the published sequences of the beta-conglycinin subunits. Comparing these cleavage sites, plus those of two others previously delineated, shows that the P1' and P4' positions always bear either a Glu or an Asp residue while the P1 position always bears either a Glu or a Gln residue. In addition, other sites from P3 to P7' are also rich in either Glu or Asp, and the whole region is predicted to be in a alpha-helix. Consistent with the observation, synthetic poly-L-Glu inhibits the Protease C1-catalysed degradation of the alpha and alpha' subunits of beta-conglycinin. Poly-L-Glu (av. M(r) = 1000) at 12.5 mM was more effective at inhibiting the reaction than poly-L-Glu (av. M(r) = 600) or poly-L-Glu (av. M(r) = 14,300) at the same concentration. Comparing large synthetic polypeptides at 12.5mM, inhibition by poly-L-Asp (av. M(r) = 15,000) is as effective as poly-L-Glu (av. M(r) = 14,300), while poly-L-Ser (av. M(r) = 15,000) had no effect at all. Poly-D-Glu (av. M(r) = 15,000) is a better inhibitor than poly-L-Glu of the same size. A serine protease of similar molecular weight as Protease C1 and also capable of catalysing the proteolysis of the alpha and alpha' subunits of beta-conglycinin to generate proteolytic intermediates of the same size has been found in mung bean.

  17. Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products.

    PubMed

    Benito, María J; Connerton, Ian F; Córdoba, Juan J

    2006-11-01

    EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.

  18. Discovery and purification of a fungal protease secreted by Bipolaris zeicola that modifies maize seed endochitinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Healthy maize seeds have two basic endochitinases, chitA and chitB, with antifungal properties. A comparison of the isoenzyme profiles of symptomatic fungal-infested maize seeds, removed at harvest from ears that we wound inoculated in the late milk stage of maturity with one of several common ear-...

  19. Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

    PubMed Central

    Sircar, Gaurab; Saha, Bodhisattwa; Mandal, Rahul Shubhra; Pandey, Naren; Saha, Sudipto; Gupta Bhattacharya, Swati

    2015-01-01

    Background Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’. Method The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. Results The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Fabrication, characterization, and enzymatic activity of fungal protease--nanogold membrane bioconjugate.

    PubMed

    Vinod, V P; Phadtare, S; Joshi, H M; Sastry, Murali; Rao, Mala

    2007-08-01

    This study describes the synthesis of a free-standing nanogold membrane by the spontaneous reduction of aqueous chloroaurate ions by the diamine molecule DAEE at a liquid-liquid interface. The free standing nanogold membrane, provides a biocompatible surface for the immobilization of proteins. F-Protease (F-Prot) was then bound to the nanogold membrane via interaction with the gold nanoparticles leading to a new class of biocatalyst. A highlight of the new biocatalyst wherein the enzyme is bound to the nanogold membrane is the ease with which separation from the reaction medium may be achieved by simple filtration. In relation to the free enzyme in solution, the F-Prot in the bioconjugate material exhibited a slightly higher biocatalytic activity and significantly enhanced pH and temperature stability. The F-Prot nanogold membrane bioconjugate material also exhibited excellent biocatalytic activity over ten successive reuse cycles.

  2. Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams.

    PubMed

    Jin, Bo; Yin, Pinghe; Ma, Yihong; Zhao, Ling

    2005-12-01

    This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94-0.97 g/g of starch or sugars associated with 4-5 g/l of fungal biomass produced, while 17-19 g/l fungal biomass with a lactic acid yield of 0.65-0.76 g/g was produced by the R. oryzae 2062 in 36-48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8-15% lactic acid yield and 10-20% fungal biomass. PMID:16208461

  3. Inhibition of citrus fungal pathogens by using lactic acid bacteria.

    PubMed

    Gerez, C L; Carbajo, M S; Rollán, G; Torres Leal, G; Font de Valdez, G

    2010-08-01

    The effect of lactic acid bacteria (LAB) on pathogenic fungi was evaluated and the metabolites involved in the antifungal effect were characterized. Penicillium digitatum (INTA 1 to INTA 7) and Geotrichum citri-aurantii (INTA 8) isolated from decayed lemon from commercial packinghouses were treated with imazalil and guazatine to obtain strains resistant to these fungicides. The most resistant strains (4 fungal strains) were selected for evaluating the antifungal activity of 33 LAB strains, among which only 8 strains gave positive results. The antifungal activity of these LAB strains was related to the production of lactic acid, acetic acid, and phenyllactic acid (PLA). A central composite design and the response surface methodology were used to evaluate the inhibitory effect of the organic acids produced by the LAB cultures. The antifungal activity of lactic acid was directly related to its concentration; however, acetic acid and PLA showed a peak of activity at 52.5 and 0.8 mM, respectively, with inhibition rates similar to those obtained with Serenade((R)) (3.0 ppm) imazalil (50 ppm) and guazatine (50 ppm). Beyond the peak of activity, a reduction in effectiveness of both acetic acid and PLA was observed. Comparing the inhibition rate of the organic acids, PLA was about 66- and 600-fold more effective than acetic acid and lactic acid, respectively. This study presents evidences on the antifungal effect of selected LAB strains and their end products. Studies are currently being undertaken to evaluate the effectiveness in preventing postharvest diseases on citrus fruits. PMID:20722936

  4. Characteristics of neutralization of acids by newly isolated fungal cells.

    PubMed

    Shiomi, Naofumi; Yasuda, Takako; Inoue, Yoko; Kusumoto, Noriko; Iwasaki, Saori; Katsuda, Tomohisa; Katoh, Shigeo

    2004-01-01

    Soil microorganisms play an important role in maintaining soil pH at levels suitable for other soil organisms. To clarify the biological neutralization mechanism in soil, we isolated soil microorganisms showing a high ability to neutralize acids and studied their characteristics. From our taxonomic study, three isolated strains were identified as filamentous fungi, namely Mucor sp., Aspergillus fumigatus, and Aureobasidium pullulans. These strains could secrete basic materials, such as ammonia, for neutralization, grow in the medium at pH 4.0 and increase the pH of the medium to approximately 8.0. These microbial cells could neutralize not only nitric acid but also sulfuric and hydrochloric acids. The strains could also grow by utilizing nitric acid as a sole nitrogen source. In the soil containing these organisms, the pH was maintained in the neutral range by the buffering action of basic materials that they secrete. These results suggest that these fungal cells are useful for protecting the soil from acidification by acid rain.

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

    SciTech Connect

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

    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.

  6. Protease inhibition by oleic acid transfer from chronic wound dressings to albumin.

    PubMed

    Edwards, J Vincent; Howley, Phyllis; Davis, Rachel; Mashchak, Andrew; Goheen, Steven C

    2007-08-01

    High elastase and cathepsin G activities have been observed in chronic wounds to inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid is a non-toxic elastase inhibitor. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of oleic acid by albumin under conditions mimicking chronic wounds. The mechanism of oleic acid uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin-oleic acid complexes under liquid equilibrium conditions revealed fully saturated albumin-oleic acid complexes with a 1:1 weight ratio of albumin:oleic acid. Liquid-solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of oleic acid per mole albumin. Comparing oleic acid formulated wound dressings for dose dependent ability to lower elastase activity, we found cotton gauze>hydrogel>hydrocolloid. In contrast, the cationic serine protease cathepsin G was inhibited by oleic acid within a narrow range of oleic acid-cotton formulations. 2% albumin was sufficient to transfer quantities of oleic acid necessary to achieve a significant elastase-lowering effect. Oleic acid bound to cotton wound dressings may have promise in the selective lowering of cationic serine protease activity useful in topical application for chronic inflammatory pathogenesis.

  7. Structural changes of oil palm empty fruit bunch (OPEFB) after fungal and phosphoric acid pretreatment.

    PubMed

    Isroi; Ishola, Mofoluwake M; Millati, Ria; Syamsiah, Siti; Cahyanto, Muhammad N; Niklasson, Claes; Taherzadeh, Mohammad J

    2012-01-01

    Oil palm empty fruit bunch (OPEFB) was pretreated using white-rot fungus Pleurotus floridanus, phosphoric acid or their combination, and the results were evaluated based on the biomass components, and its structural and morphological changes. The carbohydrate losses after fungal, phosphoric acid, and fungal followed by phosphoric acid pretreatments were 7.89%, 35.65%, and 33.77%, respectively. The pretreatments changed the hydrogen bonds of cellulose and linkages between lignin and carbohydrate, which is associated with crystallinity of cellulose of OPEFB. Lateral Order Index (LOI) of OPEFB with no pretreatment, with fungal, phosphoric acid, and fungal followed by phosphoric acid pretreatments were 2.77, 1.42, 0.67, and 0.60, respectively. Phosphoric acid pretreatment showed morphological changes of OPEFB, indicated by the damage of fibre structure into smaller particle size. The fungal-, phosphoric acid-, and fungal followed by phosphoric acid pretreatments have improved the digestibility of OPEFB's cellulose by 4, 6.3, and 7.4 folds, respectively. PMID:23247371

  8. Docking analysis of gallic acid derivatives as HIV-1 protease inhibitors.

    PubMed

    Singh, Anjali; Pal, Tapan Kumar

    2015-01-01

    HIV-1 Protease (HIV-1 PR) enzymes are essential for accurate assembly and maturation of infectious HIV retroviruses. The significant role of HIV-1 protease in viral replication has made it a potential drug target. In the recent past, phytochemical Gallic Acid (GA) derivatives have been screened for protease inhibitor activity. The present work aims to design and evaluate potential GA-based HIV-1 PR phytoinhibitors by docking approach. The ligands were prepared by ChemDraw and docking was performed in HEX software. In this present study, one of the GA analogues (GA4) emerged as a potent drug candidate for HIV-1 PR inhibition, and docking results showed it to be comparable with anti-HIV drugs, darunavir and amprenavir. The GA4 derivative provided a lead for designing more effective HIV-1 PR inhibitors.

  9. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  10. A modified method for the detection of microbial proteases on agar plates using tannic acid.

    PubMed

    Saran, Saurabh; Isar, Jasmine; Saxena, Rajendra Kumar

    2007-06-10

    In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.

  11. An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium.

    PubMed

    Kocabiyik, Semra; Ozel, Hatice

    2007-01-01

    In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.

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

    PubMed Central

    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

  13. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The 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 macrophages and MAC-T cells that 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 increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. 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. PMID:27597934

  14. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The 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 macrophages and MAC-T cells that 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 increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. 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.

  15. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    PubMed

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The 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 macrophages and MAC-T cells that 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 mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. 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. PMID:27597934

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

    PubMed

    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. PMID:27165526

  17. Diphenylarsinic acid promotes degradation of glutaminase C by mitochondrial Lon protease.

    PubMed

    Kita, Kayoko; Suzuki, Toshihide; Ochi, Takafumi

    2012-05-25

    Glutaminase C (GAC), a splicing variant of the kidney-type glutaminase (KGA) gene, is a vital mitochondrial enzyme protein that catalyzes glutamine to glutamate. Earlier studies have shown that GAC proteins in the human hepatocarcinoma cell line, HepG2, were down-regulated by diphenylarsinic acid (DPAA), but the mechanism by which DPAA induced GAC protein down-regulation remained poorly understood. Here, we showed that DPAA promoted GAC protein degradation without affecting GAC transcription and translation. Moreover, DPAA-induced GAC proteolysis was mediated by mitochondrial Lon protease. DPAA insolubilized 0.5% Triton X-100-soluble GAC protein and promoted the accumulation of insoluble GAC in Lon protease knockdown cells. DPAA destroyed the native tetrameric GAC conformation and promoted an increase in the unassembled form of GAC when DPAA was incubated with cell extracts. Decreases in the tetrameric form of GAC were observed in cells exposed to DPAA, and decreases occurred prior to a decrease in total GAC protein levels. In addition, decreases in the tetrameric form of GAC were observed independently with Lon protease. Mitochondrial heat shock protein 70 is known to be an indispensable protein that can bind to misfolded proteins, thereby supporting degradation of proteins sensitive to Lon protease. When cells were incubated with DPAA, GAC proteins that can bind with mtHsp70 increased. Interestingly, the association of mtHsp70 with GAC protein increased when the tetrameric form of GAC was reduced. These results suggest that degradation of native tetrameric GAC by DPAA may be a trigger in GAC protein degradation by Lon protease.

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

    PubMed

    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.

  19. Solid state fermentation: acid protease production in controlled CO2 and O2 environments.

    PubMed

    Villegas, E; Aubague, S; Alcantara, L; Auria, R; Revah, S

    1993-01-01

    The effect of the partial pressure of O(2) and CO(2) on the acid protease production in solid state fermentation by Aspergillus niger on wheat bran was studied. A fermentation system was used, which allowed on-line reactor measurements and continuous data acquisition of pH, temperature, gas flow, pressure drop and CO(2) production. Six paired combinations of CO(2) and O(2) concentrations were studied. The results showed a direct relationship between pressure drop, production of CO(2) and temperature increase. The pH evolution patterns were similar in all cases but different if the measurements were made on-line or on a liquid homogenate of the fermented substrate. Acid protease production was increased when the gas had 4% CO(2), (vol/vol), and it reached its highest level, a 43% increase over air, with a mixture of 4% CO(2) and 21% O(2). The protease production was strongly related to the mold metabolic activity as represented by the total CO(2) evolved.

  20. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading ac...

  1. Fungal populations in podzolic soil experimentally acidified to simulate acid rain.

    PubMed

    Bååth, E; Lundgren, B; Söderström, B

    1984-09-01

    The effect of experimental acidification on the soil microfungal community was studied in the humus layer of a coniferous forest in northern Sweden. The study was made 4 years after the last application of sulfuric acid. Fungal species composition was altered by treatments of 100 and 150 kg sulfuric acid ha(-1) each year for 6 years, yet no differences were found between the control treatment and an application of 50 kg ha(-1). The abundance ofPenicillium spinulosum andOidiodendron cf.echinulatum II increased with increasing rates of acid application, whereas only small changes were found for other isolated fungal taxa. Soil respiration rate and fluorescein diacetate (FDA)-active fungal biomass were significantly different from the control treatment at all 3 levels of acidification.

  2. A role for Lon protease in the control of the acid resistance genes of Escherichia coli.

    PubMed

    Heuveling, Johanna; Possling, Alexandra; Hengge, Regine

    2008-07-01

    Lon protease is a major protease in cellular protein quality control, but also plays an important regulatory role by degrading various naturally unstable regulators. Here, we traced additional such regulators by identifying regulons with co-ordinately altered expression in a lon mutant by genome-wide transcriptional profiling. Besides many members of the RcsA regulon (which validates our approach as RcsA is a known Lon substrate), many genes of the sigmaS-dependent general stress response were upregulated in the lon mutant. However, the lon mutation did not affect sigmaS levels nor sigmaS activity in general, suggesting specific effects of Lon on secondary regulators involved in the control of subsets of sigmaS-controlled genes. Lon-affected genes also included the major acid resistance genes (gadA, gadBC, gadE, hdeAB and hdeD), which led to the discovery that the essential acid resistance regulator GadE (whose expression is sigmaS-controlled) is degraded in vivo in a Lon-dependent manner. GadE proteolysis is constitutive as it was observed even under conditions that induce the system (i.e. at low pH or during entry into stationary phase). GadE degradation was found to rapidly terminate the acid resistance response upon shift back to neutral pH and to avoid overexpression of acid resistance genes in stationary phase.

  3. Inhibition of the keratinolytic subtilisin protease Sub3 from Microsporum canis by its propeptide (proSub3) and evaluation of the capacity of proSub3 to inhibit fungal adherence to feline epidermis.

    PubMed

    Baldo, A; Chevigné, A; Dumez, M-E; Mathy, A; Power, P; Tabart, J; Cambier, L; Galleni, M; Mignon, B

    2012-10-12

    Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats, dogs and humans. Proteolytic enzymes have been postulated to be key factors involved in the invasion of the stratum corneum and keratinized epidermal structures. Among these proteases, the secreted subtilisin protease Sub3 was found to be required for adherence of M. canis arthroconidia to feline epidermis. This protease is synthetized as a preproenzyme consisting of a signal peptide followed by the propeptide and the protease domain. In order to assess whether the enzymatic activity of Sub3 could be responsible for the role of the protease in the adherence process, we expressed and characterized the propeptide of Sub3 and demonstrated that this propeptide is a strong inhibitor of its mature enzyme. This propeptide acts as a noncompetitive inhibitor with dissociation constants, K(I) and [Formula: see text] of 170 and 130 nM respectively. When tested for its capacity to inhibit adherence of M. canis to feline epidermis using an ex vivo adherence model made of feline epidermis, the propeptide does not prevent adherence of M. canis arthroconidia because it loses its capacity to inhibit rSub3 following a direct contact with living arthroconidia, presumably through inactivation by fungal membrane-bound proteases. PMID:22633172

  4. Fungal Community Associated with Dactylopius (Hemiptera: Coccoidea: Dactylopiidae) and Its Role in Uric Acid Metabolism.

    PubMed

    Vera-Ponce de León, Arturo; Sanchez-Flores, Alejandro; Rosenblueth, Mónica; Martínez-Romero, Esperanza

    2016-01-01

    We studied fungal species associated with the carmine cochineal Dactylopius coccus and other non-domesticated Dactylopius species using culture-dependent and -independent methods. Thirty seven fungi were isolated in various culture media from insect males and females from different developmental stages and Dactylopius species. 26S rRNA genes and ITS sequences, from cultured fungal isolates revealed different species of Cryptococcus, Rhodotorula, Debaryomyces, Trametes, and Penicillium, which are genera newly associated with Dactylopius. Uric acid (UA) and uricase activity were detected in tissues extracts from different insect developmental stages. However, accumulation of high UA levels and low uricase activities were found only after antifungal treatments, suggesting an important role of fungal species in its metabolism. Additionally, uricolytic fungal isolates were identified and characterized that presumably are involved in nitrogen recycling metabolism. After metagenomic analyses from D. coccus gut and hemolymph DNA and from two published data sets, we confirmed the presence of fungal genes involved in UA catabolism, suggesting that fungi help in the nitrogen recycling process in Dactylopius by uricolysis. All these results show the importance of fungal communities in scale insects such as Dactylopius. PMID:27446001

  5. Fungal Community Associated with Dactylopius (Hemiptera: Coccoidea: Dactylopiidae) and Its Role in Uric Acid Metabolism

    PubMed Central

    Vera-Ponce de León, Arturo; Sanchez-Flores, Alejandro; Rosenblueth, Mónica; Martínez-Romero, Esperanza

    2016-01-01

    We studied fungal species associated with the carmine cochineal Dactylopius coccus and other non-domesticated Dactylopius species using culture-dependent and -independent methods. Thirty seven fungi were isolated in various culture media from insect males and females from different developmental stages and Dactylopius species. 26S rRNA genes and ITS sequences, from cultured fungal isolates revealed different species of Cryptococcus, Rhodotorula, Debaryomyces, Trametes, and Penicillium, which are genera newly associated with Dactylopius. Uric acid (UA) and uricase activity were detected in tissues extracts from different insect developmental stages. However, accumulation of high UA levels and low uricase activities were found only after antifungal treatments, suggesting an important role of fungal species in its metabolism. Additionally, uricolytic fungal isolates were identified and characterized that presumably are involved in nitrogen recycling metabolism. After metagenomic analyses from D. coccus gut and hemolymph DNA and from two published data sets, we confirmed the presence of fungal genes involved in UA catabolism, suggesting that fungi help in the nitrogen recycling process in Dactylopius by uricolysis. All these results show the importance of fungal communities in scale insects such as Dactylopius. PMID:27446001

  6. Fungal Community Associated with Dactylopius (Hemiptera: Coccoidea: Dactylopiidae) and Its Role in Uric Acid Metabolism.

    PubMed

    Vera-Ponce de León, Arturo; Sanchez-Flores, Alejandro; Rosenblueth, Mónica; Martínez-Romero, Esperanza

    2016-01-01

    We studied fungal species associated with the carmine cochineal Dactylopius coccus and other non-domesticated Dactylopius species using culture-dependent and -independent methods. Thirty seven fungi were isolated in various culture media from insect males and females from different developmental stages and Dactylopius species. 26S rRNA genes and ITS sequences, from cultured fungal isolates revealed different species of Cryptococcus, Rhodotorula, Debaryomyces, Trametes, and Penicillium, which are genera newly associated with Dactylopius. Uric acid (UA) and uricase activity were detected in tissues extracts from different insect developmental stages. However, accumulation of high UA levels and low uricase activities were found only after antifungal treatments, suggesting an important role of fungal species in its metabolism. Additionally, uricolytic fungal isolates were identified and characterized that presumably are involved in nitrogen recycling metabolism. After metagenomic analyses from D. coccus gut and hemolymph DNA and from two published data sets, we confirmed the presence of fungal genes involved in UA catabolism, suggesting that fungi help in the nitrogen recycling process in Dactylopius by uricolysis. All these results show the importance of fungal communities in scale insects such as Dactylopius.

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

    PubMed

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

    2015-11-01

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

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

    PubMed

    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.

  9. Proteolytic cleavage of human acid-sensing ion channel 1 by the serine protease matriptase.

    PubMed

    Clark, Edlira B; Jovov, Biljana; Rooj, Arun K; Fuller, Catherine M; Benos, Dale J

    2010-08-27

    Acid-sensing ion channel 1 (ASIC1) is a H(+)-gated channel of the amiloride-sensitive epithelial Na(+) channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical alphabetagammaENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems. PMID:20601429

  10. Direct fungal fermentation of lignocellulosic biomass into itaconic, fumaric, and malic acids: current and future prospects.

    PubMed

    Mondala, Andro H

    2015-04-01

    Various economic and environmental sustainability concerns as well as consumer preference for bio-based products from natural sources have paved the way for the development and expansion of biorefining technologies. These involve the conversion of renewable biomass feedstock to fuels and chemicals using biological systems as alternatives to petroleum-based products. Filamentous fungi possess an expansive portfolio of products including the multifunctional organic acids itaconic, fumaric, and malic acids that have wide-ranging current applications and potentially addressable markets as platform chemicals. However, current bioprocessing technologies for the production of these compounds are mostly based on submerged fermentation, which necessitates physicochemical pretreatment and hydrolysis of lignocellulose biomass to soluble fermentable sugars in liquid media. This review will focus on current research work on fungal production of itaconic, fumaric, and malic acids and perspectives on the potential application of solid-state fungal cultivation techniques for the consolidated hydrolysis and organic acid fermentation of lignocellulosic biomass.

  11. Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids.

    PubMed

    Rut, Wioletta; Kasperkiewicz, Paulina; Byzia, Anna; Poreba, Marcin; Groborz, Katarzyna; Drag, Marcin

    2015-04-01

    Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

  12. Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

    PubMed Central

    Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

    2015-01-01

    Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50°C) and lower stability over alkaline pH range, but better thermal stability at 60°C to 70°C and resistance to feed pelleting inactivation (80°C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing. PMID:26053048

  13. Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

    PubMed

    Yang, Wenxia; Zhang, Yuhong; Zhou, Xiaojin; Zhang, Wei; Xu, Xiaolu; Chen, Rumei; Meng, Qingchang; Yuan, Jianhua; Yang, Peilong; Yao, Bin

    2015-01-01

    Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50 °C) and lower stability over alkaline pH range, but better thermal stability at 60 °C to 70 °C and resistance to feed pelleting inactivation (80 °C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

  14. Two Proteases, Trypsin Domain-containing 1 (Tysnd1) and Peroxisomal Lon Protease (PsLon), Cooperatively Regulate Fatty Acid β-Oxidation in Peroxisomal Matrix*

    PubMed Central

    Okumoto, Kanji; Kametani, Yukari; Fujiki, Yukio

    2011-01-01

    The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon. PMID:22002062

  15. Fungal Peptaibiotics: Assessing Potential Meteoritic Amino Acid Contamination

    NASA Technical Reports Server (NTRS)

    Elsila, J. E.; Callahan, M. P.; Glavin, D. P.; Dworkin, J. P.; Bruckner, H.

    2010-01-01

    The presence of non-protein alpha-dialkyl-amino acids such as alpha-aminoisobutyric acid (alpha-A1B) and isovaline (Iva), which are relatively rare in the terrestrial biosphere, has long been used as an indication of the indigeneity of meteoritic amino acids, however, the discovery of alpha-AIB in peptides producers by a widespread group of filamentous fungi indicates the possibility of a terrestrial biotic source for the alpha-AIB observed in some meteorites. The alpha-AIB-containing peptides produced by these fungi are dubbed peptaibiotics. We measured the molecular distribution and stable carbon and nitrogen isotopic ratios for amino acids found in the total hydrolysates of four biologically synthesized peptaibiotics. We compared these aneasurenetts with those from the CM2 carbonaceous chondrite Murchison and from three Antarctic CR2 carbonaceous chondrites in order to understand the peptaibiotics as a potential source of meteoritic contamination.

  16. Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

    PubMed

    Adeniran, A J; Shoshani, I; Minuth, M; Awad, J A; Elce, J S; Johnson, R A

    1995-03-01

    We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin. PMID:7756444

  17. Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

    PubMed Central

    Klingler, Diana; Hardt, Markus

    2013-01-01

    steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides. PMID:23462971

  18. Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid.

    PubMed

    Joo, Yeon Ah; Chung, Hyunjin; Yoon, Sohyun; Park, Jong Il; Lee, Ji Eun; Myung, Cheol Hwan; Hwang, Jae Sung

    2016-09-01

    Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH₂ and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca²⁺ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH₂ and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH₂-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH₂ downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH₂ in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis. PMID:27169822

  19. Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid

    PubMed Central

    Joo, Yeon Ah; Chung, Hyunjin; Yoon, Sohyun; Park, Jong Il; Lee, Ji Eun; Myung, Cheol Hwan; Hwang, Jae Sung

    2016-01-01

    Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca2+ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis. PMID:27169822

  20. Cereal fungal infection, mycotoxins, and lactic acid bacteria mediated bioprotection: from crop farming to cereal products.

    PubMed

    Oliveira, Pedro M; Zannini, Emanuele; Arendt, Elke K

    2014-02-01

    Lactic acid bacteria (LAB) metabolites are a reliable alternative for reducing fungal infections pre-/post-harvest with additional advantages for cereal-base products which convene the food market's trend. Grain industrial use is in expansion owing to its applicability in generating functional food. The food market is directed towards functional natural food with clear health benefits for the consumer in detriment to chemical additives. The food market chain is becoming broader and more complex, which presents an ever-growing fungal threat. Toxigenic and spoilage fungi are responsible for numerous diseases and economic losses. Cereal infections may occur in the field or post-processing, along the food chain. Consequently, the investigation of LAB metabolites with antifungal activity has gained prominence in the scientific research community. LAB bioprotection retards the development of fungal diseases in the field and inhibit pathogens and spoilage fungi in food products. In addition to the health safety improvement, LAB metabolites also enhance shelf-life, organoleptic and texture qualities of cereal-base foods. This review presents an overview of the fungal impact through the cereal food chain leading to investigation on LAB antifungal compounds. Applicability of LAB in plant protection and cereal industry is discussed. Specific case studies include Fusarium head blight, malting and baking.

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

    SciTech Connect

    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

  2. Fungal hallucinogens psilocin, ibotenic acid, and muscimol: analytical methods and biologic activities.

    PubMed

    Stebelska, Katarzyna

    2013-08-01

    Psychoactive drugs of fungal origin, psilocin, ibotenic acid, and muscimol among them have been proposed for recreational use and popularized since the 1960s, XX century. Despite their well-documented neurotoxicity, they reached reputation of being safe and nonaddictive. Scientific efforts to find any medical application for these hallucinogens in psychiatry, psychotherapy, and even for religious rituals support are highly controversial. Even if they show any healing potential, their usage in psychotherapy is in some cases inadequate and may additionally harm seriously suffering patients. Hallucinogens are thought to reduce cognitive functions. However, in case of indolealkylamines, such as psilocin, some recent findings suggest their ability to improve perception and mental skills, what would motivate the consumption of "magic mushrooms." The present article offers an opportunity to find out what are the main symptoms of intoxication with mushrooms containing psilocybin/psilocin, muscimol, and ibotenic acid. The progress in analytical methods for detection of them in fungal material, food, and body fluids is reviewed. Findings on the mechanisms of their biologic activity are summarized. Additionally, therapeutic potential of these fungal psychoactive compounds and health risk associated with their abuse are discussed. PMID:23851905

  3. Fungal hallucinogens psilocin, ibotenic acid, and muscimol: analytical methods and biologic activities.

    PubMed

    Stebelska, Katarzyna

    2013-08-01

    Psychoactive drugs of fungal origin, psilocin, ibotenic acid, and muscimol among them have been proposed for recreational use and popularized since the 1960s, XX century. Despite their well-documented neurotoxicity, they reached reputation of being safe and nonaddictive. Scientific efforts to find any medical application for these hallucinogens in psychiatry, psychotherapy, and even for religious rituals support are highly controversial. Even if they show any healing potential, their usage in psychotherapy is in some cases inadequate and may additionally harm seriously suffering patients. Hallucinogens are thought to reduce cognitive functions. However, in case of indolealkylamines, such as psilocin, some recent findings suggest their ability to improve perception and mental skills, what would motivate the consumption of "magic mushrooms." The present article offers an opportunity to find out what are the main symptoms of intoxication with mushrooms containing psilocybin/psilocin, muscimol, and ibotenic acid. The progress in analytical methods for detection of them in fungal material, food, and body fluids is reviewed. Findings on the mechanisms of their biologic activity are summarized. Additionally, therapeutic potential of these fungal psychoactive compounds and health risk associated with their abuse are discussed.

  4. Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.

    PubMed

    Wolz, R L; Zeggaf, C; Stöcker, W; Zwilling, R

    1990-09-01

    Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.

  5. Biomass and organic acids in sandstone of a weathering building: Production by bacterial and fungal isolates.

    PubMed

    Palmer, R J; Siebert, J; Hirsch, P

    1991-12-01

    Ten fungal and nine bacterial strains were isolated from a weathering sandstone building. Their growth, organic acid production, and acidification capacity were assessed in culture under nutritional conditions similar to those in situ. Biomass (10-50 nmol phospholipid-PO4g(-1)) within the rock was small compared to soils. The isolated organisms were able to produce high amounts of those acids found in the sandstone, but acid production did not cause a drastic reduction in culture pH. It is suggested that the importance of acidification in microbial degradation of sandstone has been overestimated and that, under in situ pH and nutritional conditions, cation chelation by microbially produced organic acid anions may be more relevant to the weathering process.

  6. Improvement of Functional Properties of Wheat Gluten Using Acid Protease from Aspergillus usamii.

    PubMed

    Deng, Lingli; Wang, Zhaoxia; Yang, Sheng; Song, Junmei; Que, Fei; Zhang, Hui; Feng, Fengqin

    2016-01-01

    Hydrolysis parameters (temperature, E/S ratio, pH, and time) for acid protease (from Aspergillus usamii) hydrolysis of wheat gluten were optimized by response surface methodology (RSM) using emulsifying activity index (EAI) as the response factor. A temperature of 48.9°C, E/S ratio of 1.60%, pH 3.0, hydrolysis time of 2.5 h was found to be the optimum condition to obtain wheat gluten hydrolysate with higher EAI. The solubility of wheat gluten was greatly improved by hydrolysis and became independent of pH over the studied range. Enzymatic hydrolysis resulted in dramatically increase in EAI, water and oil holding capacity. Molecular weight distribution results showed that most of the peptides above 10 kDa have been hydrolyzed into smaller peptides. The results of FTIR spectra and disulfide bond (SS) and sulfhydryl (SH) content suggested that a more extensional conformation was formed after hydrolysis, which could account for the improved functional properties. PMID:27467884

  7. Improvement of Functional Properties of Wheat Gluten Using Acid Protease from Aspergillus usamii

    PubMed Central

    Deng, Lingli; Wang, Zhaoxia; Yang, Sheng; Song, Junmei; Que, Fei; Zhang, Hui; Feng, Fengqin

    2016-01-01

    Hydrolysis parameters (temperature, E/S ratio, pH, and time) for acid protease (from Aspergillus usamii) hydrolysis of wheat gluten were optimized by response surface methodology (RSM) using emulsifying activity index (EAI) as the response factor. A temperature of 48.9°C, E/S ratio of 1.60%, pH 3.0, hydrolysis time of 2.5 h was found to be the optimum condition to obtain wheat gluten hydrolysate with higher EAI. The solubility of wheat gluten was greatly improved by hydrolysis and became independent of pH over the studied range. Enzymatic hydrolysis resulted in dramatically increase in EAI, water and oil holding capacity. Molecular weight distribution results showed that most of the peptides above 10 kDa have been hydrolyzed into smaller peptides. The results of FTIR spectra and disulfide bond (SS) and sulfhydryl (SH) content suggested that a more extensional conformation was formed after hydrolysis, which could account for the improved functional properties. PMID:27467884

  8. Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis.

    PubMed

    Grąz, Marcin; Rachwał, Kamila; Zan, Radosław; Jarosz-Wilkołazka, Anna

    2016-01-01

    Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively. PMID:27337220

  9. Bioprocess development for docosahexaenoic acid from novel fungal isolate of Fusarium solani.

    PubMed

    Jini, Sugatha; Hridya, Azhikodan; Pandey, Ashok; Binod, Parameswaran

    2015-06-01

    Fungal cultures were isolated from soil samples collected from the Western Ghats regions of Kerala. Primary screening of isolated strains were done by Sudan black staining method and 15 lipid producing cultures were isolated. The fatty acid profiling of the positive strains were analyzed for docosahexaenoic acid (DHA) production. Selected oleaginous cultures were grown in submerged culture condition to study the biomass yield and poly unsaturated fatty acid, DHA production. The optimization of production process under submerged conditions was carried out using statistical experimental design and confirmation of DHA was done by GC analysis. Maximum DHA production of 150 mg/l was achieved on 4 days of incubation at submerged condition in the presence of glucose as carbon source. PMID:26155676

  10. [Coupling of protease activity and sodium loading with intestinal absorption of amino acids].

    PubMed

    Basova, N A; Markov, Iu G; Berzinia, N I

    2005-09-01

    Membrane-bound serine proteases to play a certain role in activation of sodium transport in epithelial cells. To were found explain the protease activity and sodium-dependent L-tryptophan transport across chicken small intestine interaction, four experiments were conducted. One hundred chicks were fed diets that contained 0; 0.3; 3 or 6% of supplemental NaCl and were given distillated water ad libitum. Signs of salt toxicity observed were as follows: a decreased body weight, increased heart and kidney weights, formation of secondary lysosomes in enterocytes and lymphocytes. Such chickens were in the state of negative nitrogen balance. Intestinal absorption of L-tryptophan correlated with mucosal protease activity during increased dietary sodium chloride intake. Recent in vitro and in vivo experiments indicate that enterocyte proteases may be of critical importance in activation of sodium-dependent intestinal transporters for L-tryptophan.

  11. Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    Lee, I. Russel; Yang, Liting; Sebetso, Gaseene; Allen, Rebecca; Doan, Thi H. N.; Blundell, Ross; Lui, Edmund Y. L.; Morrow, Carl A.; Fraser, James A.

    2013-01-01

    Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed. PMID:23667704

  12. Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1).

    PubMed

    Orchard, Michael G; Neuss, Judi C; Galley, Carl M S; Carr, Andrew; Porter, David W; Smith, Phillip; Scopes, David I C; Haydon, David; Vousden, Katherine; Stubberfield, Colin R; Young, Kate; Page, Martin

    2004-08-01

    The first inhibitors of fungal protein: mannosyl transferase 1 (PMT1) are described. They are based upon rhodanine-3-acetic acid and several compounds have been identified, for example, 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (5a), which inhibit Candida albicans PMT1 with IC(50)s in the range 0.2-0.5 microM. Members of the series are effective in inducing changes in morphology of C. albicans in vitro that have previously been associated with loss of the transferase activity. These compounds could serve as useful tools for studying the effects of protein O-mannosylation and its relevance in the search for novel antifungal agents. PMID:15225710

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

    PubMed

    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.

  14. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    PubMed Central

    Satpathy, Raghunath; Konkimalla, V. B.; Ratha, Jagnyeswar

    2016-01-01

    2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA), conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K) and aspartate (D) residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models. PMID:26880911

  15. Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana.

    PubMed

    Uchikoba, T; Arima, K; Yonezawa, H; Shimada, M; Kaneda, M

    2000-10-18

    A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

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

    PubMed

    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.

  17. Effect of fungal and phosphoric acid pretreatment on ethanol production from oil palm empty fruit bunches (OPEFB).

    PubMed

    Ishola, Mofoluwake M; Isroi; Taherzadeh, Mohammad J

    2014-08-01

    Oil palm empty fruit bunches (OPEFB), a lignocellulosic residue of palm oil industries was examined for ethanol production. Milled OPEFB exposed to simultaneous saccharification and fermentation (SSF) with enzymes and Saccharomyces cerevisiae resulted just in 14.5% ethanol yield compared to the theoretical yield. Therefore, chemical pretreatment with phosphoric acid, a biological pretreatment with white-rot fungus Pleurotus floridanus, and their combination were carried out on OPEFB prior to the SSF. Pretreatment with phosphoric acid, combination of both methods and just fungal pretreatment improved the digestibility of OPEFB by 24.0, 16.5 and 4.5 times, respectively. During the SSF, phosphoric acid pretreatment, combination of fungal and phosphoric acid pretreatment and just fungal pretreatment resulted in the highest 89.4%, 62.8% and 27.9% of the theoretical ethanol yield, respectively. However, the recovery of the OPEFB after the fungal pretreatment was 98.7%, which was higher than after phosphoric acid pretreatment (36.5%) and combined pretreatment (45.2%).

  18. Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases.

    PubMed Central

    Lynas, J; Walker, B

    1997-01-01

    A series of novel synthetic peptides, containing a C-terminal beta-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6x10(6) M-1. s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3x10(7) M-1.s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8x10(-5) s-1 and approx. 4.3x10(-4) s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates. PMID:9271079

  19. Crystal growth and preliminary X-ray study of glutamic acid specific serine protease from Bacillus intermedius

    NASA Astrophysics Data System (ADS)

    Kuranova, I. P.; Blagova, E. V.; Levdikov, V. M.; Rudenskaya, G. N.; Balaban, N. P.; Shakirov, E. V.

    1999-01-01

    The glutamic acid specific protease (glutamyl-endopeptidase) from Bacillus intermedius, strain 3-19, was isolated and purified using ion exchange chromatography on CM-cellulose and Mono-S FPLC column. The conditions for crystallization of the enzyme have been discussed. The crystals of enzyme were grown using hanging-drop vapor-diffusion technique. Crystals belong to the space group C2 with unit cell parameters of a=61.62 Å, b=55.84 Å, c=60.40 Å, β=117.6° X-ray diffraction data to 1.68 Å resolution were collected using synchrotron radiation (EMBL, Hamburg) and an imaging plate scanner.

  20. A high throughput method for rapid screening of chitosanase-producing fungal strain under acidic conditions.

    PubMed

    Ding, Su; Chen, Gui-Guang; Liang, Zhi-Qun; Zeng, Wei; Cao, Mu-Ming; Chen, Guo-Pin; Xie, Shu-Yu; Li, Wei

    2016-11-01

    A novel high-throughput method was established for rapid screening of a large numbers of Aspergillus fumigatus (A. fumigatus) mutants with high chitosanase production under acidic culture condition by exploiting the fact that iodine can be used as the indicator to stain chitosan but is ineffective for chitooligosaccharides. The mutant population was generated by irradiating A. fumigatus CICC 2434 with Co(60)-γ rays. Mutants were cultured on acidic plates containing colloidal chitosan and preliminary screened according to diameter of haloes formed around colonies. Then, chitosanase production of the isolates were verified by dinitrosalicylic acid assay. Lastly, molecular masses on enzymolysis products of isolated mutants were rapidly compared by aniline blue plate assay. Using this method, the mutant strain Co-8 was selected, which had chitosanase activity of 24.87 U/mL (increased by 369.2 % as compared to that of its parental strain).Taking together, the method is easy, efficient and particularly suited to rapid screen acidophilic fungal strains with high chitosanase-production. PMID:27628334

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

    PubMed

    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. Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes.

    PubMed

    Gadd, G M

    1999-01-01

    alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxido-reduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry.

  3. Distribution of tannic acid degrading microorganisms in the soil and comparative study of tannase from two fungal strains.

    PubMed

    Mondal, K C; Samanta, S; Giri, S; Pati, B R

    2001-01-01

    A quantitative survey on microbial population including tannase producing organisms have been made from different soil samples. Most of the samples harbour negligible number of tannase producers in comparison to total microbial flora. Among the tannase producers, fungal members are more frequent than bacteria. Tannase production and tannic acid degradation have been studied in two newly isolated potent fungal strains. Both the strains produce maximum tannase at their stationary phases of growth. Enzymes produced by both the strains remain active within pH 3.5-6.0 and temperature 30-60 degrees C.

  4. The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions.

    PubMed

    Durham, D R

    1990-08-01

    A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.

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

    PubMed

    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. PMID:24664515

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

    PubMed

    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.

  7. Oleaginous fungal lipid fermentation on combined acid- and alkali-pretreated corn stover hydrolysate for advanced biofuel production.

    PubMed

    Ruan, Zhenhua; Zanotti, Michael; Archer, Steven; Liao, Wei; Liu, Yan

    2014-07-01

    A combined hydrolysis process, which first mixed dilute acid- and alkali-pretreated corn stover at a 1:1 (w/w) ratio, directly followed by enzymatic saccharification without pH adjustment, has been developed in this study in order to minimize the need of neutralization, detoxification, and washing during the process of lignocellulosic biofuel production. The oleaginous fungus Mortierella isabellina was selected and applied to the combined hydrolysate as well as a synthetic medium to compare fungal lipid accumulation and biodiesel production in both shake flask and 7.5L fermentor. Fungal cultivation on combined hydrolysate exhibited comparable cell mass and lipid yield with those from synthetic medium, indicating that the integration of combined hydrolysis with oleaginous fungal lipid fermentation has great potential to improve performance of advanced lignocellulosic biofuel production.

  8. Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

    PubMed

    Chuang, L T; Thurmond, J M; Liu, J W; Kirchner, S J; Mukerji, P; Bray, T M; Huang, Y S

    2001-02-01

    Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

  9. Evolutionary origins of the multienzyme architecture of giant fungal fatty acid synthase.

    PubMed

    Bukhari, Habib S T; Jakob, Roman P; Maier, Timm

    2014-12-01

    Fungal fatty acid synthase (fFAS) is a key paradigm for the evolution of complex multienzymes. Its 48 functional domains are embedded in a matrix of scaffolding elements, which comprises almost 50% of the total sequence and determines the emergent multienzymes properties of fFAS. Catalytic domains of fFAS are derived from monofunctional bacterial enzymes, but the evolutionary origin of the scaffolding elements remains enigmatic. Here, we identify two bacterial protein families of noncanonical fatty acid biosynthesis starter enzymes and trans-acting polyketide enoyl reductases (ERs) as potential ancestors of scaffolding regions in fFAS. The architectures of both protein families are revealed by representative crystal structures of the starter enzyme FabY and DfnA-ER. In both families, a striking structural conservation of insertions to scaffolding elements in fFAS is observed, despite marginal sequence identity. The combined phylogenetic and structural data provide insights into the evolutionary origins of the complex multienzyme architecture of fFAS.

  10. T Cell Determinants Incorporating [beta]-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

    SciTech Connect

    Webb, Andrew I.; Dunstone, Michelle A.; Williamson, Nicholas A.; Price, Jason D.; Kauwe, Andreade; Chen, Weisan; Oakley, Aaron; Perlmutter, Patrick; McCluskey, James; Aguilar, Marie-Isabel; Rossjohn, Jamie; Purcell, Anthony W.

    2010-07-20

    A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of {beta}-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding {beta}-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with {beta}-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using {beta}-amino acids. We conclude that the rational incorporation of {beta}-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring {alpha}-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

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

    PubMed

    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.

  12. Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa1

    PubMed Central

    Hara, Kenichiro; Iijima, Koji; Elias, Martha K.; Seno, Satoshi; Tojima, Ichiro; Kobayashi, Takao; Kephart, Gail M.; Kurabayashi, Masahiko; Kita, Hirohito

    2014-01-01

    While type 2 immune responses to environmental antigens are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. Here we report that IL-33 and thymic stromal lymphopoietin (TSLP) were produced quickly in the lungs of naïve mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and TSLP sensitized naïve animals to an innocuous airway antigen OVA, which resulted in production of type 2 cytokines and IgE antibody and eosinophilic airway inflammation when mice were challenged with the same antigen. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naïve animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa. PMID:24663677

  13. Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets

    PubMed Central

    2012-01-01

    Background Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. Results Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. Conclusions Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary

  14. Valorisation of food waste via fungal hydrolysis and lactic acid fermentation with Lactobacillus casei Shirota.

    PubMed

    Kwan, Tsz Him; Hu, Yunzi; Lin, Carol Sze Ki

    2016-10-01

    Food waste recycling via fungal hydrolysis and lactic acid (LA) fermentation has been investigated. Hydrolysates derived from mixed food waste and bakery waste were rich in glucose (80.0-100.2gL(-1)), fructose (7.6gL(-1)) and free amino nitrogen (947-1081mgL(-1)). In the fermentation with Lactobacillus casei Shirota, 94.0gL(-1) and 82.6gL(-1) of LA were produced with productivity of 2.61gL(-1)h(-1) and 2.50gL(-1)h(-1) for mixed food waste and bakery waste hydrolysate, respectively. The yield was 0.94gg(-1) for both hydrolysates. Similar results were obtained using food waste powder hydrolysate, in which 90.1gL(-1) of LA was produced with a yield and productivity of 0.92gg(-1) and 2.50gL(-1)h(-1). The results demonstrate the feasibility of an efficient bioconversion of food waste to LA and a decentralized approach of food waste recycling in urban area.

  15. Inhibition of fungal spore adhesion by zosteric Acid as the basis for a novel, nontoxic crop protection technology.

    PubMed

    Stanley, Michele S; Callow, Maureen E; Perry, Ruth; Alberte, Randall S; Smith, Robert; Callow, James A

    2002-04-01

    ABSTRACT To explore the potential for nontoxic crop protection technologies based on the inhibition of fungal spore adhesion, we have tested the effect of synthetic zosteric acid (p-(sulfo-oxy) cinnamic acid), a naturally occurring phenolic acid in eelgrass (Zostera marina L.) plants, on spore adhesion and infection in two pathosystems: rice blast caused by Magnaporthe grisea and bean anthracnose caused by Colletotrichum lindemuthianum. We have shown that zosteric acid inhibits spore adhesion to model and host leaf surfaces and that any attached spores fail to develop appressoria, and consequently do not infect leaf cells. Low concentrations of zosteric acid that are effective in inhibiting adhesion are not toxic to either fungus or to the host. The inhibition of spore adhesion in the rice blast pathogen is fully reversible. On plants, zosteric acid reduced (rice) or delayed (bean) lesion development. These results suggest that there is potential for novel and environmentally benign crop protection technologies based on manipulating adhesion.

  16. Ubiquitin specific protease 19 involved in transcriptional repression of retinoic acid receptor by stabilizing CORO2A

    PubMed Central

    Lim, Key-Hwan; Choi, Jong-Ho; Park, Jung-Hyun; Cho, Hyeon-Ju; Park, Jang-Joon; Lee, Eung-Ji; Li, Lan; Choi, Young-Kil; Baek, Kwang-Hyun

    2016-01-01

    Deubiquitination via deubiquitinating enzymes (DUBs) has been emerged as one of the important post-translational modifications, resulting in the regulation of numerous target proteins. In this study, we screened new protein biomarkers for adipogenesis, and related studies showed that ubiquitin specific protease 19 (USP19) as a DUB is gradually decreased during adipogenesis and it regulates coronin 2A (CORO2A) as one of the components for the nuclear receptor co-repressor (NCoR) complex in some studies. The regulation of CORO2A through the deubiquitinating activity of USP19 affected the transcriptional repression activity of the retinoic acid receptor (RAR), suggesting that USP19 may be involved in the regulation of RAR-mediated adipogenesis. PMID:27129179

  17. Crystal versus solution structure of enzymes: NMR spectroscopy of a peptide boronic acid-serine protease complex in the crystalline state.

    PubMed Central

    Farr-Jones, S; Smith, S O; Kettner, C A; Griffin, R G; Bachovchin, W W

    1989-01-01

    The effectiveness of boronic acids as inhibitors of serine proteases has been widely ascribed to the ability of the boronyl group to form a tetrahedral adduct with the active-site serine that closely mimics the putative tetrahedral intermediate or transition state formed with substrates. However, recent 15N NMR studies of alpha-lytic protease (EC 3.4.21.12) in solution have shown that some boronic acids and peptide boronic acids form adducts with the active-site histidine instead of with the serine. Such histidine-boron adducts have not thus far been reported in x-ray diffraction studies of boronic acid-serine protease complexes. Here, we report an 15N NMR study of the MeOSuc-Ala-Ala-Pro-boroPhe complex of alpha-lytic protease in the crystalline state using magic-angle spinning. Previous 15N NMR studies have shown this complex involves the formation of a histidine-boron bond in solution. The 15N NMR spectra of the crystalline complex are essentially identical to those of the complex in solution, thereby showing that the structure of this complex is the same in solution and in the crystal and that both involve formation of a histidine-boron adduct. PMID:2780549

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

    PubMed

    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.

  19. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence.

    PubMed

    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.

  20. Improved protease stability of the antimicrobial peptide Pin2 substituted with D-amino acids.

    PubMed

    Carmona, G; Rodriguez, A; Juarez, D; Corzo, G; Villegas, E

    2013-08-01

    Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.

  1. Use of fungal proteases and selected sourdough lactic acid bacteria for making wheat bread with an intermediate content of gluten.

    PubMed

    Rizzello, Carlo Giuseppe; Curiel, José Antonio; Nionelli, Luana; Vincentini, Olimpia; Di Cagno, Raffaella; Silano, Marco; Gobbetti, Marco; Coda, Rossana

    2014-02-01

    This study was aimed at combining the highest degradation of gluten during wheat flour fermentation with good structural and sensory features of the related bread. As estimated by R5-ELISA, the degree of degradation of immune reactive gluten was ca. 28%. Two-dimensional electrophoresis and RP-FPLC analyses showed marked variations of the protein fractions compared to the untreated flour. The comparison was also extended to in vitro effect of the peptic/tryptic-digests towards K562 and T84 cells. The flour with the intermediate content of gluten (ICG) was used for bread making, and compared to whole gluten (WG) bread. The chemical, structural and sensory features of the ICG bread approached those of the bread made with WG flour. The protein digestibility of the ICG bread was higher than that from WG flour. Also the nutritional quality, as estimated by different indexes, was the highest for ICG bread.

  2. Firing Range Soils Yield a Diverse Array of Fungal Isolates Capable of Organic Acid Production and Pb Mineral Solubilization

    PubMed Central

    Sullivan, Tarah S.; Gottel, Neil R.; Basta, Nicholas; Jardine, Philip M.

    2012-01-01

    Anthropogenic sources of lead contamination in soils include mining and smelting activities, effluents and wastes, agricultural pesticides, domestic garbage dumps, and shooting ranges. While Pb is typically considered relatively insoluble in the soil environment, some fungi may potentially contribute to mobilization of heavy metal cations by means of secretion of low-molecular-weight organic acids (LMWOAs). We sought to better understand the potential for metal mobilization within an indigenous fungal community at an abandoned shooting range in Oak Ridge, TN, where soil Pb contamination levels ranged from 24 to >2,700 mg Pb kg dry soil−1. We utilized culture-based assays to determine organic acid secretion and Pb-carbonate dissolution of a diverse collection of soil fungal isolates derived from the site and verified isolate distribution patterns within the community by 28S rRNA gene analysis of whole soils. The fungal isolates examined included both ascomycetes and basidiomycetes that excreted high levels (up to 27 mM) of a mixture of LMWOAs, including oxalic and citric acids, and several isolates demonstrated a marked ability to dissolve Pb-carbonate at high concentrations up to 10.5 g liter−1 (18.5 mM) in laboratory assays. Fungi within the indigenous community of these highly Pb-contaminated soils are capable of LMWOA secretion at levels greater than those of well-studied model organisms, such as Aspergillus niger. Additionally, these organisms were found in high relative abundance (>1%) in some of the most heavily contaminated soils. Our data highlight the need to understand more about autochthonous fungal communities at Pb-contaminated sites and how they may impact Pb biogeochemistry, solubility, and bioavailability, thus consequently potentially impacting human and ecosystem health. PMID:22729539

  3. Firing range soils yield a diverse array of fungal isolates capable of organic acid production and Pb mineral solubilization.

    PubMed

    Sullivan, Tarah S; Gottel, Neil R; Basta, Nicholas; Jardine, Philip M; Schadt, Christopher W

    2012-09-01

    Anthropogenic sources of lead contamination in soils include mining and smelting activities, effluents and wastes, agricultural pesticides, domestic garbage dumps, and shooting ranges. While Pb is typically considered relatively insoluble in the soil environment, some fungi may potentially contribute to mobilization of heavy metal cations by means of secretion of low-molecular-weight organic acids (LMWOAs). We sought to better understand the potential for metal mobilization within an indigenous fungal community at an abandoned shooting range in Oak Ridge, TN, where soil Pb contamination levels ranged from 24 to >2,700 mg Pb kg dry soil(-1). We utilized culture-based assays to determine organic acid secretion and Pb-carbonate dissolution of a diverse collection of soil fungal isolates derived from the site and verified isolate distribution patterns within the community by 28S rRNA gene analysis of whole soils. The fungal isolates examined included both ascomycetes and basidiomycetes that excreted high levels (up to 27 mM) of a mixture of LMWOAs, including oxalic and citric acids, and several isolates demonstrated a marked ability to dissolve Pb-carbonate at high concentrations up to 10.5 g liter(-1) (18.5 mM) in laboratory assays. Fungi within the indigenous community of these highly Pb-contaminated soils are capable of LMWOA secretion at levels greater than those of well-studied model organisms, such as Aspergillus niger. Additionally, these organisms were found in high relative abundance (>1%) in some of the most heavily contaminated soils. Our data highlight the need to understand more about autochthonous fungal communities at Pb-contaminated sites and how they may impact Pb biogeochemistry, solubility, and bioavailability, thus consequently potentially impacting human and ecosystem health.

  4. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report

    SciTech Connect

    Kingsley, Mark T.

    2001-03-13

    The threat to American interests from terrorists is not limited to attacks against humans. Terrorists might seek to inflict damage to the U.S. economy by attacking our agricultural sector. Infection of commodity crops by bacterial or fungal crop pathogens could adversely impact U.S. agriculture, either directly from damage to crops or indirectly from damage to our ability to export crops suspected of contamination. Recognizing a terrorist attack against U.S. agriculture, to be able to prosecute the terrorists, is among the responsibilities of the members of Hazardous Material Response Unit (HMRU) of the Federal Bureau of Investigation (FBI). Nucleic acid analysis of plant pathogen strains by the use of polymerase chain reaction (PCR) amplification techniques is a powerful method for determining the exact identity of pathogens, as well as their possible region of origin. This type of analysis, however, requires that PCR assays be developed specific to each particular pathogen strain, and analysis protocols developed that are specific to the particular instrument used for detection. The objectives of the work described here were threefold: 1) to assess the potential terrorist threat to U.S. agricultural crops, 2) to determine whether suitable assays exist to monitor that threat, and 3) where assays are needed for priority plant pathogen threats, to modify or develop those assays for use by specialists at the HMRU. The assessment of potential threat to U.S. commodity crops and the availability of assays for those threats were described in detail in the Technical Requirements Document (9) and will be summarized in this report. This report addresses development of specific assays identified in the Technical Requirements Document, and offers recommendations for future development to ensure that HMRU specialists will be prepared with the PCR assays they need to protect against the threat of economic terrorism.

  5. Nucleic Acid-Based Detection and Identification of Bacterial and Fungal Plant Pathogens - Final Report

    SciTech Connect

    Kingsley, Mark T

    2001-03-13

    The threat to American interests from terrorists is not limited to attacks against humans. Terrorists might seek to inflict damage to the U.S. economy by attacking our agricultural sector. Infection of commodity crops by bacterial or fungal crop pathogens could adversely impact U.S. agriculture, either directly from damage to crops or indirectly from damage to our ability to export crops suspected of contamination. Recognizing a terrorist attack against U.S. agriculture, to be able to prosecute the terrorists, is among the responsibilities of the members of Hazardous Material Response Unit (HMRU) of the Federal Bureau of Investigation (FBI). Nucleic acid analysis of plant pathogen strains by the use of polymerase chain reaction (PCR) amplification techniques is a powerful method for determining the exact identity of pathogens, as well as their possible region of origin. This type of analysis, however, requires that PCR assays be developed specific to each particular pathogen strain, an d analysis protocols developed that are specific to the particular instrument used for detection. The objectives of the work described here were threefold: (1) to assess the potential terrorist threat to U.S. agricultural crops, (2) to determine whether suitable assays exist to monitor that threat, and (3) where assays are needed for priority plant pathogen threats, to modify or develop those assays for use by specialists at the HMRU. The assessment of potential threat to U.S. commodity crops and the availability of assays for those threats were described in detail in the Technical Requirements Document (9) and will be summarized in this report. This report addresses development of specific assays identified in the Technical Requirements Document, and offers recommendations for future development to ensure that HMRU specialists will be prepared with the PCR assays they need to protect against the threat of economic terrorism.

  6. Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen.

    PubMed

    Matsui, T; Sakurai, Y; Fujimura, Y; Hayashi, I; Oh-Ishi, S; Suzuki, M; Hamako, J; Yamamoto, Y; Yamazaki, J; Kinoshita, M; Titani, K

    1998-03-15

    We have isolated a serine protease, halystase, from Agkistrodon halys blomhoffii venom by chromatography on DEAE-Sepharose, heparin-Sepharose and Q-Sepharose columns, and have determined the complete amino acid sequence by Edman degradation and by mass spectral analysis of peptides generated by enzymatic and chemical cleavage. The 238-residue sequence of halystase, containing N-linked carbohydrates (about 13%) at two sites showed significant similarity to other thrombin-like snake venom serine proteases (66-72%), mammalian tissue kallikrein (42%) and thrombin (26%). Halystase contained the tentative catalytic triad of His43, Asp88 and Ser184 common to all serine proteases and Asp178 in the primary substrate-binding site. Although halystase contained an RGD sequence at residues 181-183, it did not inhibit platelet aggregation induced by ADP or collagen. It hydrolyzed most efficiently a tissue-kallikrein substrate, prolylphenylalanylarginyl-4-methyl-coumaryl-7-amide, and released bradykinin from bovine kininogen. Halystase did not coagulate human plasma, but it cleaved the fibrinogen B beta chain at the carboxyl side of Arg42 and cleaved slowly the fibrogen A alpha chain. Fibrinogen thus treated gradually became insensitive to thrombin. The proteolytic activity was inhibited with diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride or leupeptin. These results indicate that halystase is a serine protease structurally similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves fibrinogen at sites different from thrombin without inducing fibrin clotting, and hydrolyzes kininogen to produce bradykinin, resulting in the reduction of blood pressure.

  7. Jasmonic acid signalling mediates resistance of the wild tobacco Nicotiana attenuata to its native Fusarium, but not Alternaria, fungal pathogens.

    PubMed

    Luu, Van Thi; Schuck, Stefan; Kim, Sang-Gyu; Weinhold, Arne; Baldwin, Ian T

    2015-03-01

    We recently characterized a highly dynamic fungal disease outbreak in native populations of Nicotiana attenuata in the southwestern United States. Here, we explore how phytohormone signalling contributes to the observed disease dynamics. Single inoculation with three native Fusarium and Alternaria fungal pathogens, isolated from diseased plants growing in native populations, resulted in disease symptoms characteristic for each pathogen species. While Alternaria sp.-infected plants displayed fewer symptoms and recovered, Fusarium spp.-infected plants became chlorotic and frequently spontaneously wilted. Jasmonic acid (JA) and salicylic acid (SA) levels were differentially induced after Fusarium or Alternaria infection. Transgenic N. attenuata lines silenced in JA production or JA conjugation to isoleucine (JA-Ile), but not in JA perception, were highly susceptible to infection by F. brachygibbosum Utah 4, indicating that products derived from the JA-Ile biosynthetic pathway, but not their perception, is associated with increased Fusarium resistance. Infection assays using ov-nahG plants which were silenced in pathogen-induced SA accumulations revealed that SA may increase N. attenuata's resistance to Fusarium infection but not to Alternaria. Taken together, we propose that the dynamics of fungal disease symptoms among plants in native populations may be explained by a complex interplay of phytohormone responses to attack by multiple pathogens.

  8. Fungal biotransformation of chlorogenic and caffeic acids by Fusarium graminearum: New insights in the contribution of phenolic acids to resistance to deoxynivalenol accumulation in cereals.

    PubMed

    Gauthier, Léa; Bonnin-Verdal, Marie-Noelle; Marchegay, Gisèle; Pinson-Gadais, Laetitia; Ducos, Christine; Richard-Forget, Florence; Atanasova-Penichon, Vessela

    2016-03-16

    Fusarium Head Blight and Gibberella Ear Rot, mainly caused by the fungi Fusarium graminearum and Fusarium culmorum, are two of the most devastating diseases of small-grain cereals and maize. In addition to yield loss, these diseases frequently result in contamination of kernels with toxic type B trichothecenes. The potential involvement of chlorogenic acid in cereal resistance to Fusarium Head Blight and Gibberella Ear Rot and to trichothecene accumulation was the focus of this study. The effects of chlorogenic acid and one of its hydrolyzed products, caffeic acid, on fungal growth and type B trichothecenes biosynthesis were studied using concentrations close to physiological amounts quantified in kernels and a set of F. graminearum and F. culmorum strains. Both chlorogenic and caffeic acids negatively impact fungal growth and mycotoxin production, with caffeic acid being significantly more toxic. Inhibitory efficiencies of both phenolic acids were strain-dependent. To further investigate the antifungal and anti "mycotoxin" effect of chlorogenic and caffeic acids, the metabolic fate of these two phenolic acids was characterized in supplemented F. graminearum broths. For the first time, our results demonstrated the ability of F. graminearum to degrade chlorogenic acid into caffeic, hydroxychlorogenic and protocatechuic acids and caffeic acid into protocatechuic and hydroxycaffeic acids. Some of these metabolic products can contribute to the inhibitory efficiency of chlorogenic acid that, therefore, can be compared as a "pro-drug". As a whole, our data corroborate the contribution of chlorogenic acid to the chemical defense that cereals employ to counteract F. graminearum and its production of mycotoxins. PMID:26812586

  9. Fungal biotransformation of chlorogenic and caffeic acids by Fusarium graminearum: New insights in the contribution of phenolic acids to resistance to deoxynivalenol accumulation in cereals.

    PubMed

    Gauthier, Léa; Bonnin-Verdal, Marie-Noelle; Marchegay, Gisèle; Pinson-Gadais, Laetitia; Ducos, Christine; Richard-Forget, Florence; Atanasova-Penichon, Vessela

    2016-03-16

    Fusarium Head Blight and Gibberella Ear Rot, mainly caused by the fungi Fusarium graminearum and Fusarium culmorum, are two of the most devastating diseases of small-grain cereals and maize. In addition to yield loss, these diseases frequently result in contamination of kernels with toxic type B trichothecenes. The potential involvement of chlorogenic acid in cereal resistance to Fusarium Head Blight and Gibberella Ear Rot and to trichothecene accumulation was the focus of this study. The effects of chlorogenic acid and one of its hydrolyzed products, caffeic acid, on fungal growth and type B trichothecenes biosynthesis were studied using concentrations close to physiological amounts quantified in kernels and a set of F. graminearum and F. culmorum strains. Both chlorogenic and caffeic acids negatively impact fungal growth and mycotoxin production, with caffeic acid being significantly more toxic. Inhibitory efficiencies of both phenolic acids were strain-dependent. To further investigate the antifungal and anti "mycotoxin" effect of chlorogenic and caffeic acids, the metabolic fate of these two phenolic acids was characterized in supplemented F. graminearum broths. For the first time, our results demonstrated the ability of F. graminearum to degrade chlorogenic acid into caffeic, hydroxychlorogenic and protocatechuic acids and caffeic acid into protocatechuic and hydroxycaffeic acids. Some of these metabolic products can contribute to the inhibitory efficiency of chlorogenic acid that, therefore, can be compared as a "pro-drug". As a whole, our data corroborate the contribution of chlorogenic acid to the chemical defense that cereals employ to counteract F. graminearum and its production of mycotoxins.

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

    NASA Astrophysics Data System (ADS)

    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 ( 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 of poTRY, 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.

  11. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

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

    PubMed Central

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

    2016-01-01

    Nitrated fatty acids (NFAs), endogenous products of nonenzymatic reactions of NO-derived reactive nitrogen species with unsaturated fatty acids, exhibit substantial anti-inflammatory activities. They are both reversible electrophiles and peroxisome proliferator-activated receptor γ (PPARγ) agonists, but the physiological implications of their electrophilic activity are poorly understood. We tested their effects on inflammatory and emphysema-related biomarkers in alveolar macrophages (AMs) of smoke-exposed mice. NFA (10-nitro-oleic acid or 12-nitrolinoleic acid) treatment downregulated expression and activity of the inflammatory transcription factor NF-κB while upregulating those of PPARγ. It also downregulated production of inflammatory cytokines and chemokines and of the protease cathepsin S (Cat S), a key mediator of emphysematous septal destruction. Cat S downregulation was accompanied by decreased AM elastolytic activity, a major mechanism of septal destruction. NFAs downregulated both Cat S expression and activity in AMs of wild-type mice, but only inhibited its activity in AMs of PPARγ knockout mice, pointing to a PPARγ-independent mechanism of enzyme inhibition. We hypothesized that this mechanism was electrophilic S-alkylation of target Cat S cysteines, and found that NFAs bind directly to Cat S following treatment of intact AMs and, as suggested by in silico modeling and calculation of relevant parameters, elicit S-alkylation of Cys25 when incubated with purified Cat S. These results demonstrate that NFAs’ electrophilic activity, in addition to their role as PPARγ agonists, underlies their protective effects in chronic obstructive pulmonary disease (COPD) and support their therapeutic potential in this disease. PMID:27119365

  13. Supermarket Proteases.

    ERIC Educational Resources Information Center

    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)

  14. Mutualistic fungal endophytes produce phytohormones and organic acids that promote japonica rice plant growth under prolonged heat stress*

    PubMed Central

    Waqas, Muhammad; Khan, Abdul Latif; Shahzad, Raheem; Ullah, Ihsan; Khan, Abdur Rahim; Lee, In-Jung

    2015-01-01

    This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The japonica rice was grown in controlled chamber conditions with and without P. formosus LWL1 under no stress (NS) and prolonged heat stress (HS) conditions. Endophytic association under NS and HS conditions significantly improved plant growth attributes, such as plant height, fresh weight, dry weight, and chlorophyll content. Furthermore, P. formosus LWL1 protected the rice plants from HS compared with controls, indicated by the lower endogenous level of stress-signaling compounds such as abscisic acid (25.71%) and jasmonic acid (34.57%) and the increase in total protein content (18.76%–33.22%). Such fungal endophytes may be helpful for sustainable crop production under high environmental temperatures. PMID:26642184

  15. Mutualistic fungal endophytes produce phytohormones and organic acids that promote japonica rice plant growth under prolonged heat stress.

    PubMed

    Waqas, Muhammad; Khan, Abdul Latif; Shahzad, Raheem; Ullah, Ihsan; Khan, Abdur Rahim; Lee, In-Jung

    2015-12-01

    This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The japonica rice was grown in controlled chamber conditions with and without P. formosus LWL1 under no stress (NS) and prolonged heat stress (HS) conditions. Endophytic association under NS and HS conditions significantly improved plant growth attributes, such as plant height, fresh weight, dry weight, and chlorophyll content. Furthermore, P. formosus LWL1 protected the rice plants from HS compared with controls, indicated by the lower endogenous level of stress-signaling compounds such as abscisic acid (25.71%) and jasmonic acid (34.57%) and the increase in total protein content (18.76%-33.22%). Such fungal endophytes may be helpful for sustainable crop production under high environmental temperatures.

  16. Mutualistic fungal endophytes produce phytohormones and organic acids that promote japonica rice plant growth under prolonged heat stress.

    PubMed

    Waqas, Muhammad; Khan, Abdul Latif; Shahzad, Raheem; Ullah, Ihsan; Khan, Abdur Rahim; Lee, In-Jung

    2015-12-01

    This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The japonica rice was grown in controlled chamber conditions with and without P. formosus LWL1 under no stress (NS) and prolonged heat stress (HS) conditions. Endophytic association under NS and HS conditions significantly improved plant growth attributes, such as plant height, fresh weight, dry weight, and chlorophyll content. Furthermore, P. formosus LWL1 protected the rice plants from HS compared with controls, indicated by the lower endogenous level of stress-signaling compounds such as abscisic acid (25.71%) and jasmonic acid (34.57%) and the increase in total protein content (18.76%-33.22%). Such fungal endophytes may be helpful for sustainable crop production under high environmental temperatures. PMID:26642184

  17. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease

    SciTech Connect

    Kane, S.E.; Troen, B.R.; Gal, S.; Ueda, K.; Pastan, I.; Gottesman, M.M.

    1988-08-01

    Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, the authors transfected cloned genes for mouse or human MEP into mouse MIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes were also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

  18. Potential of protease inhibitor in 3-nitropropionic acid induced Huntington's disease like symptoms: mitochondrial dysfunction and neurodegeneration.

    PubMed

    Hariharan, Ashwini; Shetty, Shruthi; Shirole, Trupti; Jagtap, Aarti G

    2014-12-01

    Huntington's disease (HD) is a genetic, neurodegenerative disorder mainly characterized by motor dysfunction, cognitive decline and psychiatric disturbances. 3-Nitropropionic acid (3-NP) is an inhibitor of succinate dehydrogenase (Complex II) of the mitochondrial respiratory chain, which thereby reduces production of ATP. It induces neurotoxicity by causing striatal degeneration, energy deficit and oxidative stress. Angiotensin converting enzyme (ACE) is an important protease in the renin angiotensin system (RAS) responsible for the conversion of Angiotensin I to Angiotensin II. Angiotensin-II stimulates mitochondrial oxidant release leading to depression of energy metabolism. ACE inhibitors have shown promise in disorders like stress, anxiety, and depression in addition to showing beneficial effects in cognitive disorders like Alzheimer's. Angiotensin-II inhibition enhances energy production by lowering mitochondrial oxidant production, and hence protects mitochondrial structure. Trandolapril is a centrally active ACE inhibitor. 3-NP administered systematically (20mg/kg, i.p) for 4 days consecutively induced HD like symptoms - loss of body weight, neurobehavioral alterations like memory dysfunction (elevated plus maze, Morris water maze performance), Hind-limb impairment (Narrow beam test), motor incoordination (locomotor activity). Biochemical studies on brain tissue showed increased lipid peroxidation, nitrite levels and acetylcholinesterase activity along with decreased levels of reduced glutathione, catalase activity. Mitochondrial enzyme complex activities (I, II, IV and MTT assay) were found to be significantly lowered in brain mitochondria. Administration of Trandolapril (4 and 6 mg/kg, p.o) daily for 12 days showed significant improvement in body weight, neurobehavioral parameters, oxidative stress and mitochondrial enzyme activities in rat brain. These findings were further confirmed by histopathological studies which showed improvement in 3-NP induced

  19. Potential of protease inhibitor in 3-nitropropionic acid induced Huntington's disease like symptoms: mitochondrial dysfunction and neurodegeneration.

    PubMed

    Hariharan, Ashwini; Shetty, Shruthi; Shirole, Trupti; Jagtap, Aarti G

    2014-12-01

    Huntington's disease (HD) is a genetic, neurodegenerative disorder mainly characterized by motor dysfunction, cognitive decline and psychiatric disturbances. 3-Nitropropionic acid (3-NP) is an inhibitor of succinate dehydrogenase (Complex II) of the mitochondrial respiratory chain, which thereby reduces production of ATP. It induces neurotoxicity by causing striatal degeneration, energy deficit and oxidative stress. Angiotensin converting enzyme (ACE) is an important protease in the renin angiotensin system (RAS) responsible for the conversion of Angiotensin I to Angiotensin II. Angiotensin-II stimulates mitochondrial oxidant release leading to depression of energy metabolism. ACE inhibitors have shown promise in disorders like stress, anxiety, and depression in addition to showing beneficial effects in cognitive disorders like Alzheimer's. Angiotensin-II inhibition enhances energy production by lowering mitochondrial oxidant production, and hence protects mitochondrial structure. Trandolapril is a centrally active ACE inhibitor. 3-NP administered systematically (20mg/kg, i.p) for 4 days consecutively induced HD like symptoms - loss of body weight, neurobehavioral alterations like memory dysfunction (elevated plus maze, Morris water maze performance), Hind-limb impairment (Narrow beam test), motor incoordination (locomotor activity). Biochemical studies on brain tissue showed increased lipid peroxidation, nitrite levels and acetylcholinesterase activity along with decreased levels of reduced glutathione, catalase activity. Mitochondrial enzyme complex activities (I, II, IV and MTT assay) were found to be significantly lowered in brain mitochondria. Administration of Trandolapril (4 and 6 mg/kg, p.o) daily for 12 days showed significant improvement in body weight, neurobehavioral parameters, oxidative stress and mitochondrial enzyme activities in rat brain. These findings were further confirmed by histopathological studies which showed improvement in 3-NP induced

  20. Candida glabrata Persistence in Mice Does Not Depend on Host Immunosuppression and Is Unaffected by Fungal Amino Acid Auxotrophy▿

    PubMed Central

    Jacobsen, I. D.; Brunke, S.; Seider, K.; Schwarzmüller, T.; Firon, A.; d'Enfért, C.; Kuchler, K.; Hube, B.

    2010-01-01

    Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts. PMID:20008535

  1. Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

    PubMed Central

    Jones, Stephen K.; Clarke, Starlynn C.; Craik, Charles S.

    2015-01-01

    ABSTRACT Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a “barrier” and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target. PMID:26604258

  2. Structural Insight into Serine Protease Rv3671c that Protects M. tuberculosis from Oxidative and Acidic Stress

    SciTech Connect

    Biswas, Tapan; Small, Jennifer; Vandal, Omar; Odaira, Toshiko; Deng, Haiteng; Ehrt, Sabine; Tsodikov, Oleg V.

    2010-11-15

    Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo.

  3. Acyl-carrier protein - Phosphopantetheinyltransferase partnerships in fungal fatty acid synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis of fatty acids is an essential primary metabolic process for energy storage and cellular structural integrity. Assembly of saturated fatty acids is achieved by fatty acid synthases (FASs) that combine acetyl- and malonyl-CoAs by repetitive decarboxylative Claisen condensations with su...

  4. Extracellular proteases of Trichoderma species. A review.

    PubMed

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

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed. PMID:16003937

  5. Contrasting Patterns of Diterpene Acid Induction by Red Pine and White Spruce to Simulated Bark Beetle Attack, and Interspecific Differences in Sensitivity Among Fungal Associates.

    PubMed

    Mason, Charles J; Klepzig, Kier D; Kopper, Brian J; Kersten, Philip J; Illman, Barbara L; Raffa, Kenneth F

    2015-06-01

    Conifers possess a suite of physiochemical defenses that protect their subcortical tissues from bark beetle - fungal complexes. These defenses include rapid induction of terpenoids and phenolics at the site of attack. Studies of the distribution, induction, and bioactivity of conifer terpenoids have focused heavily on monoterpenes. We assessed induction of diterpene acids in white spruce (Picea glauca) and red pine (Pinus resinosa) to fungal associates of two bark beetles, and the responses of four spruce beetle (Dendroctonus rufipennis)-associated fungi to three diterpene acids. Constitutive phloem contents differed between species, in that red pine had extremely low concentrations of diterpene acids, whereas white spruce had substantial constitutive levels. Induction differed quantitatively. Both red pine and white spruce exhibited marked increases, but red pine underwent greater increases and achieved higher concentrations than white spruce. Induction also differed qualitatively in that red pine showed lower diversity and fewer compositional changes during induction than white spruce. In red pine,fungal inoculation accompanying wounding elicited greater increases than wounding alone, but in white spruce total concentrations were higher following wounding alone. Spruce beetle fungal symbiont growth varied among species and compounds. Some diterpenes elicited both stimulatory and inhibitory effects on fungi, depending on concentration. All four fungi exhibited higher tolerances compared to those associated with pine bark beetles in previous studies. Variation in tolerances to, and potentially metabolism of, diterpene acids by symbionts may reflect differences in constitutive levels between spruce and pine, and partially explain differences in concentrations achieved during induction.

  6. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  7. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  8. Cleavage of peptide bonds bearing ionizable amino acids at P{sub 1} by serine proteases with hydrophobic S{sub 1} pocket

    SciTech Connect

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

    2010-10-01

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

  9. Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase, albumin, catalase, red cell alpha-esterase-3, group-specific component, and protease inhibitor.

    PubMed

    Tan, S G; Barker, J S; Selvaraj, O S; Mukherjee, T K; Wong, Y F

    1993-06-01

    We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell alpha-esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.

  10. Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana.

    PubMed

    Yonezawa, H; Uchikoba, T; Arima, K; Shimada, M; Kaneda, M

    1999-07-01

    A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.

  11. Polymerization of pentachlorophenol and ferulic acid by fungal extracellular lignin-degrading enzymes.

    PubMed Central

    Rüttimann-Johnson, C; Lamar, R T

    1996-01-01

    High-molecular-weight polymers were produced by a crude concentrated supernatant from ligninolytic Phanerochaete chrysosporium cultures in a reaction mixture containing pentachlorophenol and a humic acid precursor (ferulic acid) in the presence of a detergent and H2O2. Pure manganese peroxidase, lignin peroxidase, and laccase were also shown to catalyze the reaction. PMID:8967777

  12. The natural diyne-furan fatty acid EV-086 is an inhibitor of fungal delta-9 fatty acid desaturation with efficacy in a model of skin dermatophytosis.

    PubMed

    Knechtle, Philipp; Diefenbacher, Melanie; Greve, Katrine B V; Brianza, Federico; Folly, Christophe; Heider, Harald; Lone, Museer A; Long, Lisa; Meyer, Jean-Philippe; Roussel, Patrick; Ghannoum, Mahmoud A; Schneiter, Roger; Sorensen, Alexandra S

    2014-01-01

    Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida, Aspergillus, and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae, depending on the delta-9 fatty acid desaturase OLE1. Delta-9 unsaturated fatty acids-but not saturated fatty acids-antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086-phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses.

  13. Fungal Tests

    MedlinePlus

    ... effectiveness of treatment. For many superficial skin and yeast infections, a clinical examination of the affected person ... the chemical solution dissolves non-fungal elements; reveals yeast cells and fungal hyphae (branching filaments) on a ...

  14. Fungal arthritis

    MedlinePlus

    Mycotic arthritis; Infectious arthritis - fungal ... Marquez J, Espinoza LR. Infectious arthritis II: mycobacterial, brucellar, fungal, and parasitic arthritis. In: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH, eds. Rheumatology . ...

  15. MNL1 regulates weak acid-induced stress responses of the fungal pathogen Candida albicans.

    PubMed

    Ramsdale, Mark; Selway, Laura; Stead, David; Walker, Jan; Yin, Zhikang; Nicholls, Susan M; Crowe, Jonathan; Sheils, Emma M; Brown, Alistair J P

    2008-10-01

    MNL1, the Candida albicans homologue of an orphan Msn2-like gene (YER130c in Saccharomyces cerevisiae) has no known function. Here we report that MNL1 regulates weak acid stress responses. Deletion of MNL1 prevents the long-term adaptation of C. albicans cells to weak acid stresses and compromises their global transcriptional response under these conditions. The promoters of Mnl1-dependent genes contain a novel STRE-like element (SLE) that imposes Mnl1-dependent, weak acid stress-induced transcription upon a lacZ reporter in C. albicans. The SLE (HHYYCCCCTTYTY) is related to the Nrg1 response element (NRE) element recognized by the transcriptional repressor Nrg1. Deletion of NRG1 partially restores the ability of C. albicans mnl1 cells to adapt to weak acid stress, indicating that Mnl1 and Nrg1 act antagonistically to regulate this response. Molecular, microarray, and proteomic analyses revealed that Mnl1-dependent adaptation does not occur in cells exposed to proapoptotic or pronecrotic doses of weak acid, suggesting that Ras-pathway activation might suppress the Mnl1-dependent weak acid response in dying cells. Our work defines a role for this YER130c orthologue in stress adaptation and cell death. PMID:18653474

  16. Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus.

    PubMed

    Stentelaire, C; Lesage-Meessen, L; Oddou, J; Bernard, O; Bastin, G; Ceccaldi, B C; Asther, M

    2000-01-01

    The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l. PMID:16232733

  17. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety. PMID:19244904

  18. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  19. Nestedness in Arbuscular Mycorrhizal Fungal Communities along Soil pH Gradients in Early Primary Succession: Acid-Tolerant Fungi Are pH Generalists

    PubMed Central

    Kawahara, Ai; An, Gi-Hong; Miyakawa, Sachie; Sonoda, Jun

    2016-01-01

    Soil acidity is a major constraint on plant productivity. Arbuscular mycorrhizal (AM) fungi support plant colonization in acidic soil, but soil acidity also constrains fungal growth and diversity. Fungi in extreme environments generally evolve towards specialists, suggesting that AM fungi in acidic soil are acidic-soil specialists. In our previous surveys, however, some AM fungi detected in strongly acidic soils could also be detected in a soil with moderate pH, which raised a hypothesis that the fungi in acidic soils are pH generalists. To test the hypothesis, we conducted a pH-manipulation experiment and also analyzed AM fungal distribution along a pH gradient in the field using a synthesized dataset of the previous and recent surveys. Rhizosphere soils of the generalist plant Miscanthus sinensis were collected both from a neutral soil and an acidic soil, and M. sinensis seedlings were grown at three different pH. For the analysis of field communities, rhizosphere soils of M. sinensis were collected from six field sites across Japan, which covered a soil pH range of 3.0–7.4, and subjected to soil trap culture. AM fungal community compositions were determined based on LSU rDNA sequences. In the pH-manipulation experiment the acidification of medium had a significant impact on the compositions of the community from the neutral soil, but the neutralization of the medium had no effect on those of the community from the acidic soil. Furthermore, the communities in lower -pH soils were subsets of (nested in) those in higher-pH soils. In the field communities a significant nestedness pattern was observed along the pH gradient. These observations suggest that the fungi in strongly acidic soils are pH generalists that occur not only in acidic soil but also in wide ranges of soil pH. Nestedness in AM fungal community along pH gradients may have important implications for plant community resilience and early primary succession after disturbance in acidic soils. PMID

  20. Influence of calcium on fungal growth, hyphal morphology and citric acid production in Aspergillus niger.

    PubMed

    Pera, L M; Callieri, D A

    1997-01-01

    Addition of 0.5 g/L CaCl2 to the fermentation medium lowered the final biomass dry mass by 35% and increased the uptake of phosphate and sucrose, and the production of citric acid by 15, 35 and 50%, respectively. In a medium deprived of Ca2+ the microorganism displayed both a pelleted and a filamentous form of growth, the hyphae being scarcely branched, without bulbous cells. An addition of Ca2+ induced a pelleted form of growth, highly branched hyphae and numerous bulbous cells. Bulbous cells growing in the presence of Ca2+ exhibited cell walls composed of laminated layers, and featured vesicles associated with the wall and/or the cell membrane, containing numerous inclusions. The cytotoxic effect of high concentrations of citric acid in the medium as well as an increase of the activity of N-acetyl-beta-D-glucosaminidase, a lytic enzyme, might be involved in these morphological changes.

  1. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    PubMed

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  2. Cryptococcus neoformans can utilize the bacterial melanin precursor homogentisic acid for fungal melanogenesis.

    PubMed

    Frases, Susana; Salazar, Angela; Dadachova, Ekaterina; Casadevall, Arturo

    2007-01-01

    Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin "ghost"-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.

  3. Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis▿

    PubMed Central

    Frases, Susana; Salazar, Angela; Dadachova, Ekaterina; Casadevall, Arturo

    2007-01-01

    Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment. PMID:17098915

  4. From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

    PubMed

    Olmedo, Andrés; Aranda, Carmen; Del Río, José C; Kiebist, Jan; Scheibner, Katrin; Martínez, Angel T; Gutiérrez, Ana

    2016-09-26

    A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.

  5. From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

    PubMed

    Olmedo, Andrés; Aranda, Carmen; Del Río, José C; Kiebist, Jan; Scheibner, Katrin; Martínez, Angel T; Gutiérrez, Ana

    2016-09-26

    A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids. PMID:27573441

  6. Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease

    PubMed Central

    De Marco, Janice L; Felix, Carlos Roberto

    2002-01-01

    Background Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall. Results A protease produced by the Trichoderma harzianum isolate 1051 was purified to homogeneity by precipitation with ammonium sulfate followed by hydrophobic chromatography. The molecular mass of this protease as determined by SDS-polyacrylamide gel electrophoresis was about 18.8 kDa. Its N-terminal amino acid sequence shares no homology with any other protease. The purified enzyme substantially affected the cell wall of the phytopathogen C. perniciosa. Western-blotting analysis showed that the enzyme was present in the culture supernatant 24 h after the Trichoderma started to grow in casein-containing liquid medium. Conclusions The capacity of the Trichoderma harzianum protease to hydrolyze the cell wall of C. perniciosa indicates that this enzyme may be actually involved in the antagonistic process between the two fungi. This fact strongly suggest that hydrolytic enzyme over-producing transgenic fungi may show superior biocontrol capacity. PMID:11835696

  7. A Conserved Hydrogen-Bonding Network of P2 bis-Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

    PubMed Central

    Yedidi, Ravikiran S.; Garimella, Harisha; Aoki, Manabu; Aoki-Ogata, Hiromi; Desai, Darshan V.; Chang, Simon B.; Davis, David A.; Fyvie, W. Sean; Kaufman, Joshua D.; Smith, David W.; Das, Debananda; Wingfield, Paul T.; Maeda, Kenji; Ghosh, Arun K.

    2014-01-01

    In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRVRP20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRVRP20. PMID:24752271

  8. Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

    PubMed Central

    Lee, Cheng-Siang; Taib, Nur Aishah Mohd; Ashrafzadeh, Ali; Fadzli, Farhana; Harun, Faizah; Rahmat, Kartini; Hoong, See Mee; Abdul-Rahman, Puteri Shafinaz; Hashim, Onn Haji

    2016-01-01

    Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations. PMID:26890881

  9. Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer.

    PubMed

    Lee, Cheng-Siang; Taib, Nur Aishah Mohd; Ashrafzadeh, Ali; Fadzli, Farhana; Harun, Faizah; Rahmat, Kartini; Hoong, See Mee; Abdul-Rahman, Puteri Shafinaz; Hashim, Onn Haji

    2016-01-01

    Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15-3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.

  10. Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer.

    PubMed

    Lee, Cheng-Siang; Taib, Nur Aishah Mohd; Ashrafzadeh, Ali; Fadzli, Farhana; Harun, Faizah; Rahmat, Kartini; Hoong, See Mee; Abdul-Rahman, Puteri Shafinaz; Hashim, Onn Haji

    2016-01-01

    Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15-3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations. PMID:26890881

  11. 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.

    PubMed

    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. PMID:26362088

  12. Optimizing the saccharification of sugar cane bagasse using dilute phosphoric acid followed by fungal cellulases.

    PubMed

    Geddes, C C; Peterson, J J; Roslander, C; Zacchi, G; Mullinnix, M T; Shanmugam, K T; Ingram, L O

    2010-03-01

    A low level of phosphoric acid (1% w/w on dry bagasse basis, 160 degrees C and above, 10 min) was shown to effectively hydrolyze the hemicellulose in sugar cane bagasse into monomers with minimal side reactions and to serve as an effective pre-treatment for the enzymatic hydrolysis of cellulose. Up to 45% of the remaining water-insoluble solids (WIS) was digested to sugar monomers by a low concentration of Biocellulase W (0.5 filter paper unit/gWIS) supplemented with beta-glucosidase, although much higher levels of cellulase (100-fold) were required for complete hydrolysis. After neutralization and nutrient addition, phosphoric acid syrups of hemicellulose sugars were fermented by ethanologenic Escherichia coli LY160 without further purification. Fermentation of these syrups was preceded by a lag that increased with increased pre-treatment temperature. Further improvements in organisms and optimization of steam treatments may allow the co-fermentation of sugars derived from hemicellulose and cellulose, eliminating need for liquid-solid separation, sugar purification, and separate fermentations.

  13. Cloning, expression, and characterization of a milk-clotting aspartic protease gene (Po-Asp) from Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Chen, Liguo; Tan, Qi; Shang, Xiaodong; Ma, Aimin

    2014-02-01

    An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3' portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68-80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.

  14. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; 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 metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  15. Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry

    SciTech Connect

    Sagaram, Uma S.; El-Mounadi, Kaoutar; Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghoottama; Smith, Thomas J.; Shah, Dilip

    2013-12-04

    A highly conserved plant defensin MtDef4 potently inhibits the growth of a filamentous fungus Fusarium graminearum. MtDef4 is internalized by cells of F. graminearum. To determine its mechanism of fungal cell entry and antifungal action, NMR solution structure of MtDef4 has been determined. The analysis of its structure has revealed a positively charged patch on the surface of the protein consisting of arginine residues in its γ-core signature, a major determinant of the antifungal activity of MtDef4. Here, we report functional analysis of the RGFRRR motif of the γ-core signature of MtDef4. The replacement of RGFRRR to AAAARR or to RGFRAA not only abolishes fungal cell entry but also results in loss of the antifungal activity of MtDef4. MtDef4 binds strongly to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Mutations of RGFRRR which abolish fungal cell entry of MtDef4 also impair its binding to PA. Our results suggest that RGFRRR motif is a translocation signal for entry of MtDef4 into fungal cells and that this positively charged motif likely mediates interaction of this defensin with PA as part of its antifungal action.

  16. Fungal Endocarditis

    PubMed Central

    Yuan, Shi-Min

    2016-01-01

    Fungal endocarditis is a rare and fatal condition. The Candida and Aspergillus species are the two most common etiologic fungi found responsible for fungal endocarditis. Fever and changing heart murmur are the most common clinical manifestations. Some patients may have a fever of unknown origin as the onset symptom. The diagnosis of fungal endocarditis is challenging, and diagnosis of prosthetic valve fungal endocarditis is extremely difficult. The optimum antifungal therapy still remains debatable. Treating Candida endocarditis can be difficult because the Candida species can form biofilms on native and prosthetic heart valves. Combined treatment appears superior to monotherapy. Combination of antifungal therapy and surgical debridement might bring about better prognosis. PMID:27737409

  17. Fungal Sinusitis.

    PubMed

    Raz, Eytan; Win, William; Hagiwara, Mari; Lui, Yvonne W; Cohen, Benjamin; Fatterpekar, Girish M

    2015-11-01

    Fungal sinusitis is characterized into invasive and noninvasive forms. The invasive variety is further classified into acute, chronic and granulomatous forms; and the noninvasive variety into fungus ball and allergic fungal sinusitis. Each of these different forms has a unique radiologic appearance. The clinicopathologic and corresponding radiologic spectrum and differences in treatment strategies of fungal sinusitis make it an important diagnosis for clinicians and radiologists to always consider. This is particularly true of invasive fungal sinusitis, which typically affects immuno compromised patients and is associated with significant morbidity and mortality. Early diagnosis allows initiation of appropriate treatment strategies resulting in favorable outcome.

  18. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

    PubMed Central

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

  19. Advances in protease engineering for laundry detergents.

    PubMed

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

    2015-12-25

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

  20. Structure and Mechanism of Action of the Protease That Degrades Small, Acid-Soluble Spore Proteins during Germination of Spores of Bacillus Species

    PubMed Central

    Nessi, Claudio; Jedrzejas, Mark J.; Setlow, Peter

    1998-01-01

    The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P46) removes an ∼15-kDa C-terminal domain generating a 30-kDa species (P30) which is stable against further digestion. While P30 is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P46 to the active 41-kDa form of GPR (P41). Trypsin treatment of P41 cleaves the same bond in the C-terminal part of the protein as is cleaved in the P46→P30 conversion. While the ∼29-kDa species generated by trypsin treatment of P41 is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P41 which is 13°C lower than that for P46 and the unfolding of P41 at significantly lower concentrations of guanidine hydrochloride than for P46, are further evidence for a difference in tertiary structure between P46 and P41, with P46 presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P46 and P41. The removal of ∼30% of P46 or P41 without significant loss in enzyme activity localized GPR’s catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P41 by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease. PMID:9748439

  1. Crystal structures of Bacillus subtilis Lon protease.

    PubMed

    Duman, Ramona E; Löwe, Jan

    2010-08-27

    Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.

  2. Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance, stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots.

    PubMed

    Vahabi, Khabat; Camehl, Iris; Sherameti, Irena; Oelmüller, Ralf

    2013-11-01

    The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H2O2/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe2+ -dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.

  3. Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development.

    PubMed

    Stack, Colin M; Lowther, Jonathan; Cunningham, Eithne; Donnelly, Sheila; Gardiner, Donald L; Trenholme, Katharine R; Skinner-Adams, Tina S; Teuscher, Franka; Grembecka, Jolanta; Mucha, Artur; Kafarski, Pawel; Lua, Linda; Bell, Angus; Dalton, John P

    2007-01-19

    Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs. PMID:17107951

  4. A protease-insensitive feruloyl esterase from China Holstein cow rumen metagenomic library: expression, characterization, and utilization in ferulic acid release from wheat straw.

    PubMed

    Cheng, Fansheng; Sheng, Jiping; Cai, Ting; Jin, Jian; Liu, Wanzhen; Lin, Yanmei; Du, Yongxin; Zhang, Maoqiu; Shen, Lin

    2012-03-14

    A metagenomic library of China Holstein cow rumen microbes was constructed and screened for novel gene cluster. A novel feruloyl esterase (FAE) gene was identified with a length of 789 bp and encoded a protein displaying 56% identity to known esterase sequences. The gene was functionally expressed in Escherichia coli BL21 (DE3), and the total molecular weight of the recombined protein was 32.4 kDa. The purified enzyme showed a broad specificity against the four methyl esters of hydroxycinnamic acids and high activity (259.5 U/mg) to methyl ferulate at optimum conditions (pH 8.0, 40 °C). High thermal and pH stability were also observed. Moreover, the enzyme showed broad resistance to proteases. FAE-SH1 can enhance the release of ferulic acid from wheat straw with cellulase, β-1,4-endoxylanase, β-1,3-glucanase, and pectase. These features suggest FAE-SH1 as a good candidate to enhance biomass degradation and improve the health effects of food and forage.

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

    SciTech Connect

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

    1982-08-15

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

  6. Activity of hydrolytic enzymes in fungi isolated from diabetic pregnant women: is there any relationship between fungal alkaline and acid phosphatase activity and glycemic control?

    PubMed

    Nowakowska, Dorota; Kurnatowska, Alicja; Stray-Pedersen, Babill; Wilczyński, Jan

    2004-06-01

    Ability to respond to environmental changes and secretion of hydrolases are considered to be important for Candida virulence. In this study we determined and compared the activities of 19 different hydrolases of the fungal strains isolated from diabetic and non-diabetic pregnant women. We also looked for the presence of a relationship between hydrolase activities and glycemic control, and, furthermore, evaluated the influence of gestational age on the activity of hydrolases. Mycological examinations were performed for 119 diabetic pregnant women: 47 with diabetes mellitus type I (DM), 72 with gestational diabetes (GDM), and for 132 healthy women (CON). Samples were collected from the vagina, rectum and oral cavity and cultured on Sabouraud media. The fungal hydrolase activities were evaluated using the API ZYM test (bioMerieux). For the 19 different fungal hydrolases tested, 13 activities were present in the isolated fungal strains. The activity of alkaline phosphatase (ALP) in vaginal strains (p=0.028) and acid phosphatase (ACP) in strains from the vagina (p=0.006) and rectum (p=0.049) was significantly lower in DM than in GDM and CON women. In conclusion, we describe for the first time that fungi isolated from pregnant diabetic women have lower activity of both phosphatases compared to fungi isolated from healthy women. Furthermore, similar differences of mean ALP and ACP activities were observed in the course of pregnancy in strains from the vagina and rectum of DM and CON women. However, strains from DM had lower activity at each stage of pregnancy. The highest activity of ALP and ACP was detected at the beginning, then declined, and had the lowest values between the 24(th) and 33(rd) week of gestation. After that period the activity of both phosphatases increased.

  7. Negligible contribution from roots to soil-borne phospholipid fatty acid fungal biomarkers 18:2ω6,9 and 18:1ω9.

    PubMed

    Kaiser, Christina; Frank, Alexander; Wild, Birgit; Koranda, Marianne; Richter, Andreas

    2010-09-01

    The phospholipid fatty acid biomarkers 18:1ω9, 18:2ω6,9 and 18:3ω3,6,9 are commonly used as fungal biomarkers in soils. They have, however, also been found to occur in plant tissues, such as roots. Thus, the use of these PLFAs as fungal biomarkers in sieved soil, which may still contain small remains of roots, has been questioned. We used data from a recent beech tree girdling experiment to calculate the contribution of roots to these biomarkers and were able to demonstrate that not more than 0.61% of 18:1ω9 and 18:2ω6,9 in sieved soil samples originated from roots (but 4% of 18:3ω3,6,9). Additionally, the abundance of the biomarker 18:2ω6,9 in the soil was found to be highly correlated to ectomycorrhizal root colonization, which further corroborates its fungal origin. PLFA biomarkers were substantially reduced in vital roots from girdled trees compared to roots of control trees (by up to 76%), indicating that the major part of PLFAs measured in roots may actually originate from ectomycorrhizal fungi growing inside the roots. We calculated, that even a near to 50% reduction in fine root biomass - as observed in the girdling treatment - accounted for only 0.8% of the measured decrease of 18:2ω6,9. Our results demonstrate that both 18:1ω9 and 18:2ω6,9 are suitable biomarkers for detecting fungal dynamics in soils and that especially 18:2ω6,9 is a reliable biomarker to study mycorrhizal dynamics in beech forests.

  8. Fungal allergens.

    PubMed Central

    Horner, W E; Helbling, A; Salvaggio, J E; Lehrer, S B

    1995-01-01

    Airborne fungal spores occur widely and often in far greater concentrations than pollen grains. Immunoglobulin E-specific antigens (allergens) on airborne fungal spores induce type I hypersensitivity (allergic) respiratory reactions in sensitized atopic subjects, causing rhinitis and/or asthma. The prevalence of respiratory allergy to fungi is imprecisely known but is estimated at 20 to 30% of atopic (allergy-predisposed) individuals or up to 6% of the general population. Diagnosis and immunotherapy of allergy to fungi require well-characterized or standardized extracts that contain the relevant allergen(s) of the appropriate fungus. Production of standardized extracts is difficult since fungal extracts are complex mixtures and a variety of fungi are allergenic. Thus, the currently available extracts are largely nonstandardized, even uncharacterized, crude extracts. Recent significant progress in isolating and characterizing relevant fungal allergens is summarized in the present review. Particularly, some allergens from the genera Alternaria, Aspergillus, and Cladosporium are now thoroughly characterized, and allergens from several other genera, including some basidiomycetes, have also been purified. The availability of these extracts will facilitate definitive studies of fungal allergy prevalence and immunotherapy efficacy as well as enhance both the diagnosis and therapy of fungal allergy. PMID:7621398

  9. Ruminal bacterial, archaeal, and fungal diversity of dairy cows in response to ingestion of lauric or myristic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this experiment, part of a larger study, was to investigate changes in rumen bacterial, archaeal, and fungal diversity in cows with normal and reduced protozoal populations. In the main study, 6 lactating dairy cows were dosed intraruminally with 240 g/cow per day of stearic (contr...

  10. Fungal nail infection

    MedlinePlus

    Nails - fungal infection; Onychomycosis; Infection - fungal - nails; Tinea unguium ... hair, nails, and outer skin layers. Common fungal infections include: Athlete's foot Jock itch Ringworm on the ...

  11. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  12. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination.

    PubMed Central

    Sussman, M D; Setlow, P

    1991-01-01

    The gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination, has been cloned from Bacillus megaterium and Bacillus subtilis, and its nucleotide sequence has been determined. Use of a translational gpr-lacZ fusion showed that the B. subtilis gpr gene was expressed primarily, if not exclusively, in the forespore compartment of the sporulating cell, with expression taking place approximately 1 h before expression of glucose dehydrogenase and ssp genes. gpr-lacZ expression was abolished in spoIIAC (sigF) and spoIIIE mutants but was reduced only approximately 50% in a spoIIIG (sigG) mutant. However, the kinetics of the initial approximately 50% of gpr-lacZ expression were unaltered in a spoIIIG mutant. The in vivo transcription start site of gpr has been identified and found to be identical to the in vitro start site on this gene with either E sigma F or E sigma G. Induction of sigma G synthesis in vivo turned on gpr-lacZ expression in parallel with synthesis of glucose dehydrogenase. These data are consistent with gpr transcription during sporulation first by E sigma F and then by E sigma G. Images PMID:1840582

  13. Cr(VI) reduction by gluconolactone and hydrogen peroxide, the reaction products of fungal glucose oxidase: Cooperative interaction with organic acids in the biotransformation of Cr(VI).

    PubMed

    Romo-Rodríguez, Pamela; Acevedo-Aguilar, Francisco Javier; Lopez-Torres, Adolfo; Wrobel, Kazimierz; Wrobel, Katarzyna; Gutiérrez-Corona, J Félix

    2015-09-01

    The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI).

  14. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  15. Fungal Infections

    MedlinePlus

    ... it, you'll be saying bye-bye to fungi (say: FUN-guy). What Is a Fungal Infection? Fungi , the word for more than one fungus, can ... but of course, they're not!). Because the fungi that cause tinea (ringworm) live on different parts ...

  16. In vitro and ex vivo anti-human immunodeficiency virus (HIV) activities of a new water-soluble HIV protease inhibitor, R-87366, containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid.

    PubMed

    Komai, T; Yagi, R; Suzuki-Sunagawa, H; Sakurai, M; Higashida, S; Sugano, M; Handa, H; Mohri, H; Yasuoka, A; Oka, S; Yabe, Y; Nishigaki, T; Kimura, S; Shimada, K

    1997-02-01

    In a series of compounds containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA), a transitionstate mimetic, R-87366:(2S,3S)-3-[N-(quinoxaline-2-carbonyl)-L-asparaginyl]amino- 2-hydroxy-4-phenylbutanoyl-L-proline tert-butylamide, was found to be a potent human immunodeficiency virus protease inhibitor (Ki value was 11 nM) and anti-HIV agent (IC90 value was 0.5 microM for HIV-1IIIB acutely infected cells) with moderate water-solubility (4.2 mg/ml at 25 degrees C). The compound was also active in chronically infected Molt-4/HIV-1IIIB cells, and inhibited the proteolytic processing of p55 into p17, suggesting that its anti-HIV activity was derived from HIV protease inhibition. The compound showed more potent activity (IC90 value was 0.03-0.25 microM) against clinical isolates of HIV in 5 out of 6 patients examined with varying clinical status in an ex vivo assay. One isolate, however, from the sixth patient, was less sensitive to R-87366 (IC90 value was 0.5 microM). In experiments with this strain, R-87366 showed comparatively low efficacy in acutely infected peripheral blood mononuclear cell (PBMC). This result suggests that the diversity of sensitivity shown in the ex vivo assay could be caused by the viral property itself. As a result of the determination of nucleic acid sequences in the clinical isolates, some amino acids were found to be substituted in the protease region, in contrast to the HIV-1 clade B consensus sequence, and some of them have been reported to contribute to the susceptibility of HIV protease inhibitors.

  17. Rapid real-time nucleic Acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections.

    PubMed

    Zhao, Yanan; Park, Steven; Kreiswirth, Barry N; Ginocchio, Christine C; Veyret, Raphaël; Laayoun, Ali; Troesch, Alain; Perlin, David S

    2009-07-01

    Bloodstream infections (BSIs) are a significant cause of morbidity and mortality. Successful patient outcomes are diminished by a failure to rapidly diagnose these infections and initiate appropriate therapy. A rapid and reliable diagnostic platform of high sensitivity is needed for the management of patients with BSIs. The combination of an RNA-dependent nucleic acid sequence-based amplification and molecular beacon (NASBA-MB) detection system in multiplex format was developed to rapidly detect medically important BSI organisms. Probes and primers representing pan-gram-negative, pan-gram-positive, pan-fungal, pan-Candida, and pan-Aspergillus organisms were established utilizing 16S and 28S rRNA targets for bacteria and fungi, respectively. Two multiplex panels were developed to rapidly discriminate bacterial or fungal infections at the subkingdom/genus level with a sensitivity of 1 to 50 genomes. A clinical study was performed to evaluate the accuracy of this platform by evaluating 570 clinical samples from a tertiary-care hospital group using blood bottle samples. The sensitivity, specificity, and Youden's index values for pan-gram-positive detection and pan-gram-negative detection were 99.7%, 100%, 0.997 and 98.6%, 95.9%, 0.945, respectively. The positive predictive values (PPV) and the negative predictive values (NPV) for these two probes were 100, 90.7, and 99.4, 99.4, respectively. Pan-fungal and pan-Candida probes showed 100% sensitivity, specificity, PPV, and NPV, and the pan-Aspergillus probe showed 100% NPV. Robust signals were observed for all probes in the multiplex panels, with signal detection in <15 min. The multiplex real-time NASBA-MB assay provides a valuable platform for the rapid and specific diagnosis of bloodstream pathogens, and reliable pathogen identification and characterization can be obtained in under 3 h.

  18. Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis.

    PubMed

    Rodrigues, M L; Rozental, S; Couceiro, J N; Angluster, J; Alviano, C S; Travassos, L R

    1997-12-01

    Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis.

  19. Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis.

    PubMed Central

    Rodrigues, M L; Rozental, S; Couceiro, J N; Angluster, J; Alviano, C S; Travassos, L R

    1997-01-01

    Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis. PMID:9393779

  20. Proteases as therapeutics

    PubMed Central

    Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

    2015-01-01

    Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

  1. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

    PubMed Central

    Jashni, Mansoor Karimi; Mehrabi, Rahim; Collemare, Jérôme; Mesarich, Carl H.; de Wit, Pierre J. G. M.

    2015-01-01

    Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles. PMID:26284100

  2. Electrocatalytic oxidation of phytohormone salicylic acid at copper nanoparticles-modified gold electrode and its detection in oilseed rape infected with fungal pathogen Sclerotinia sclerotiorum.

    PubMed

    Wang, Zhan; Wei, Fang; Liu, Sheng-Yi; Xu, Qiao; Huang, Jun-Yan; Dong, Xu-Yan; Yu, Jiu-Hong; Yang, Qin; Zhao, Yuan-Di; Chen, Hong

    2010-01-15

    Salicylic acid (SA) is a biological substance that acts as a phytohormone and plays an important role in signal transduction in plants. It is important to accurately and sensitively detect SA levels. A gold electrode modified with copper nanoparticles was used to assay the electrocatalytic oxidation of salicylic acid. It was found that the electrochemical behavior of salicylic acid was greatly improved at copper nanoparticles, indicating that anodic oxidation could be catalyzed at copper nanoparticles. And the pH had remarkable effect on the electrochemical process, a very well-defined oxidation peak appeared at pH 13.3 (0.2M NaOH). The kinetics parameters of this process were calculated and the heterogeneous electron transfer rate constant (k) was determined to be 1.34x10(-3)cms(-1), and (1-alpha)n(alpha) was 1.22. The gold electrode modified with copper nanoparticles could detect SA at a higher sensitivity than common electrodes. The electrode was used to detect the SA levels in oilseed rape infected with the fungal pathogen Sclerotinia sclerotiorum. The results showed that the SA concentration reached a maximum during the 10th-25th hours after infection. This result was very similar to that determined by HPLC, indicating that the gold electrodes modified with copper nanoparticles could be used as salicylic acid sensors.

  3. Fungal proteinaceous compounds with multiple biological activities.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Chan, Yau Sang; Dan, Xiuli; Pan, Wenliang; Wang, Hexiang; Guan, Suzhen; Chan, Ki; Ye, Xiuyun; Liu, Fang; Xia, Lixin; Chan, Wai Yee

    2016-08-01

    Fungi comprise organisms like molds, yeasts and mushrooms. They have been used as food or medicine for a long time. A large number of fungal proteins or peptides with diverse biological activities are considered as antibacterial, antifungal, antiviral and anticancer agents. They encompass proteases, ribosome inactivating proteins, defensins, hemolysins, lectins, laccases, ribonucleases, immunomodulatory proteins, and polysaccharopeptides. The target of the present review is to update the status of the various bioactivities of these fungal proteins and peptides and discuss their therapeutic potential. PMID:27338574

  4. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  5. Fungal keratitis

    PubMed Central

    Tuli, Sonal S

    2011-01-01

    Clinical question: What is the most appropriate management of fungal keratitis? Results: Traditionally, topical Natamycin is the most commonly used medication for filamentous fungi while Amphotericin B is most commonly used for yeast. Voriconazole is rapidly becoming the drug of choice for all fungal keratitis because of its wide spectrum of coverage and increased penetration into the cornea. Implementation: Repeated debridement of the ulcer is recommended for the penetration of topical medications. While small, peripheral ulcers may be treated in the community, larger or central ulcers, especially if associated with signs suggestive of anterior chamber penetration should be referred to a tertiary center. Prolonged therapy for approximately four weeks is usually necessary. PMID:21468333

  6. Fungal Diagnostics

    PubMed Central

    Kozel, Thomas R.; Wickes, Brian

    2014-01-01

    Early diagnosis of fungal infection is critical to effective treatment. There are many impediments to diagnosis such as a diminishing number of clinical mycologists, cost, time to result, and requirements for sensitivity and specificity. In addition, fungal diagnostics must meet the contrasting needs presented by the increasing diversity of fungi found in association with the use of immunosuppressive agents in countries with high levels of medical care and the need for diagnostics in resource-limited countries where large numbers of opportunistic infections occur in patients with AIDS. Traditional approaches to diagnosis include direct microscopic examination of clinical samples, histopathology, culture, and serology. Emerging technologies include molecular diagnostics and antigen detection in clinical samples. Innovative new technologies that use molecular and immunoassay platforms have the potential to meet the needs of both resource-rich and resource-limited clinical environments. PMID:24692193

  7. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

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

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

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

    SciTech Connect

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

    1987-08-01

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

  9. Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

    PubMed Central

    Hayashida, Shinsaku; Teramoto, Yuji

    1986-01-01

    Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme. Images PMID:16347204

  10. Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production

    PubMed Central

    El-Tayeb, T.S.; Abdelhafez, A.A.; Ali, S.H.; Ramadan, E.M.

    2012-01-01

    This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120 min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker’s yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker’s yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained. PMID:24031984

  11. Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production.

    PubMed

    El-Tayeb, T S; Abdelhafez, A A; Ali, S H; Ramadan, E M

    2012-10-01

    This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120 min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker's yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker's yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained. PMID:24031984

  12. Molecular Cloning and Characterization of a Subtilisin-Like Serine Protease Gene (Pr1) from the Medicinal Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

    PubMed

    Shi, Ping; Lu, Zenghui; He, Yuanchuan; Chen, Shijiang; Yan, Jun; Li, Junhong; Zhang, Xiaobing

    2015-01-01

    In the Chinese caterpillar mushroom Ophiocordyceps sinensis, a subtislin-like serine protease (Pr1) is one of the most important enzymes for its infection activity against insect cuticles. The Pr1 gene was isolated from the valuable Chinese medicinal fungus O. sinensis using rapid amplification of 5' and 3' complementary DNA ends. The 2079-bp full-length complementary DNA sequence containing the 1605-bp predicted open reading frame of the Pr1 gene was obtained (GenBank accession no. KF836756). The open reading frame encodes a protein comprising 534 amino acids. Protein sequence multiple alignment analysis revealed high homology with 16 other subtilisin serine proteases and exhibited the highly conserved catalytic domain (D195, H227, and S393). We also constructed a phylogenetic tree in this study. Further molecular studies are needed to elucidate the mechanisms of fungal infection. PMID:26853964

  13. Cellular responses induced in vitro by pestheic acid, a fungal metabolite, in a gastric adenocarcinoma cell line (PG100).

    PubMed

    Sousa, J M C; Matos, L A; Alcântara, D F A; Ribeiro, H F; Santos, L S; Oliveira, M N; Brito-Junior, L C; Khayat, A S; Guimarães, A C; Cunha, L A; Burbano, R R; Bahia, M O

    2013-01-01

    There is a constant search for new cancer treatments that are less aggressive and economically affordable. In this context, natural products extracted from plants, fungi, and microorganisms are of great interest. Pestheic acid, or dihidromaldoxin, is a chlorinated diphenylic ether extracted from the phytopathogenic fungus Pestalotiopsis guepinii (Amphisphaeriaceae). We assessed the cytotoxic, cytostatic, and genotoxic effects of pestheic acid in a gastric adenocarcinoma cell line (PG100). A decrease in clonogenic survival was observed. Pestheic acid also induced significant increases in both micronucleus and nucleoplasmic bridge frequency. However, we did not observe changes in cell cycle kinetics or apoptosis induction. Reactive oxygen species induced by diphenylic ethers may explain the genotoxicity and mutagenicity of pestheic acid. The absence of repair checkpoints that we observed is probably due to the fact that the PG100 cell line lacks the TP53 gene, which is common in gastric cancers. Even though pestheic acid has had a clear cytotoxic effect, the minimal inhibitory concentration was high, which shows that pestheic acid is not an active anticancer compound under these conditions. PMID:24114206

  14. Fungal bioconversion of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP).

    PubMed

    Vroumsia, T; Steiman, R; Seigle-Murandi, F; Benoit-Guyod, J-L

    2005-09-01

    Ninety strains of fungi from the collection of our mycology laboratory were tested in Galzy and Slonimski (GS) synthetic liquid medium for their ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its by-product, 2,4-dichlorophenol (2,4-DCP) at 100 mg l(-1), each. Evolution of the amounts of each chemical in the culture media was monitored by HPLC. After 5 days of cultivation, the best results were obtained with Aspergillus penicilloides and Mortierella isabellina for 2,4-D and with Chrysosporium pannorum and Mucor genevensis for 2,4-DCP. The data collected seemed to prove, on one hand, that the strains responses varied with the taxonomic groups and the chemicals tested, and, on the other hand, that 2,4-D was less accessible to fungal degradation than 2,4-DCP. In each case, kinetics studies with the two most efficient strains revealed that there was a lag phase of 1 day before the onset of 2,4-D degradation, whereas there was none during 2,4-DCP degradation. Moreover, 2,4-DCP was detected transiently during 2,4-D degradation. Finally, M. isabellina improved its degradation potential in Tartaric Acid (TA) medium relative to GS and Malt Extract (ME) media.

  15. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    PubMed Central

    Inácio, Fabíola Dorneles; Ferreira, Roselene Oliveira; de Araujo, Caroline Aparecida Vaz; Brugnari, Tatiane; Castoldi, Rafael; Peralta, Rosane Marina; de Souza, Cristina Giatti Marques

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. PMID:26180792

  16. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

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

    2016-09-01

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

  17. Structural Analysis of Fungal Cerebrosides

    PubMed Central

    Barreto-Bergter, Eliana; Sassaki, Guilherme L.; de Souza, Lauro M.

    2011-01-01

    Of the ceramide monohexosides (CMHs), gluco- and galactosyl-ceramides are the main neutral glycosphingolipids expressed in fungal cells. Their structural determination is greatly dependent on the use of mass spectrometric techniques, including fast atom bombardment-mass spectrometry, electrospray ionization, and energy collision-induced dissociation mass spectrometry. Nuclear magnetic resonance has also been used successfully. Such a combination of techniques, combined with classical analytical separation, such as high-performance thin layer chromatography and column chromatography, has led to the structural elucidation of a great number of fungal CMHs. The structure of fungal CMH is conserved among fungal species and consists of a glucose or galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to hydroxylated fatty acids, most commonly having 16 or 18 carbon atoms and unsaturation between C-3 and C-4. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. Fungal cerebrosides were also characterized as antigenic molecules directly or indirectly involved in cell growth or differentiation in Schizophyllum commune, Cryptococcus neoformans, Pseudallescheria boydii, Candida albicans, Aspergillus nidulans, Aspergillus fumigatus, and Colletotrichum gloeosporioides. Besides classical techniques for cerebroside (CMH) analysis, we now describe new approaches, combining conventional thin layer chromatography and mass spectrometry, as well as emerging technologies for subcellular localization and distribution of glycosphingolipids by secondary ion mass spectrometry and imaging matrix-assisted laser desorption ionization time-of-flight. PMID:22164155

  18. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    PubMed

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae.

  19. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    PubMed

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae. PMID:26851403

  20. Production of hydroxy-fatty acid derivatives from waste oil by Escherichia coli cells producing fungal cytochrome P450foxy.

    PubMed

    Kitazume, Tatsuya; Yamazaki, Yuya; Matsuyama, Shigeru; Shoun, Hirofumi; Takaya, Naoki

    2008-07-01

    Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7-16 carbon atoms to their omega-1 to omega-3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to omega-1 to omega-3 hydroxy decanoic acids in the order of omega-1>omega-2>omega-3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of omega-1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry.

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

    PubMed

    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.

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

    PubMed

    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. PMID:22888800

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

    PubMed

    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.

  4. Protease activities of Candida spp. isolated from otitis externa: preliminary result.

    PubMed

    Arsović, N A; Banko, A V; Dimitrijević, M V; Djordjević, V Z; Milovanović, J P; Arsenijević, V A

    2009-01-01

    Otomycosis is a fungal infection of the ear predominantly caused by Candida and Aspergillus spp. The possible virulence factors of Candida spp. are enzymes, such as proteases, phospholipases, phosphatases and esterase. According to our knowledge, protease production in Candida strains isolated from patients with otomycosis has not been investigated. The present study was aimed at determining in vitro protease activity in 8 strains of Candida spp. (C. parapsilosis, C. famata, C. guilliermondii and C. albicans) isolated from children with otomycosis. A majority of isolated strains 7/8 (87.5%) were protease positive. The protease activity ranged from Pz 0.61 to 0.78. Further investigation is necessary to clarify the contribution of protease production to Candida virulence associated with otomycosis.

  5. Endophytic fungal association via gibberellins and indole acetic acid can improve plant growth under abiotic stress: an example of Paecilomyces formosus LHL10

    PubMed Central

    2012-01-01

    Background Endophytic fungi are little known for exogenous secretion of phytohormones and mitigation of salinity stress, which is a major limiting factor for agriculture production worldwide. Current study was designed to isolate phytohormone producing endophytic fungus from the roots of cucumber plant and identify its role in plant growth and stress tolerance under saline conditions. Results We isolated nine endophytic fungi from the roots of cucumber plant and screened their culture filtrates (CF) on gibberellins (GAs) deficient mutant rice cultivar Waito-C and normal GAs biosynthesis rice cultivar Dongjin-byeo. The CF of a fungal isolate CSH-6H significantly increased the growth of Waito-C and Dongjin-byeo seedlings as compared to control. Analysis of the CF showed presence of GAs (GA1, GA3, GA4, GA8, GA9, GA12, GA20 and GA24) and indole acetic acid. The endophyte CSH-6H was identified as a strain of Paecilomyces formosus LHL10 on the basis of phylogenetic analysis of ITS sequence similarity. Under salinity stress, P. formosus inoculation significantly enhanced cucumber shoot length and allied growth characteristics as compared to non-inoculated control plants. The hypha of P. formosus was also observed in the cortical and pericycle regions of the host-plant roots and was successfully re-isolated using PCR techniques. P. formosus association counteracted the adverse effects of salinity by accumulating proline and antioxidants and maintaining plant water potential. Thus the electrolytic leakage and membrane damage to the cucumber plants was reduced in the association of endophyte. Reduced content of stress responsive abscisic acid suggest lesser stress convened to endophyte-associated plants. On contrary, elevated endogenous GAs (GA3, GA4, GA12 and GA20) contents in endophyte-associated cucumber plants evidenced salinity stress modulation. Conclusion The results reveal that mutualistic interactions of phytohormones secreting endophytic fungi can ameliorate host

  6. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    PubMed

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

  7. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    PubMed

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. PMID:26264818

  8. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    PubMed

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

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

  9. Burdock fructooligosaccharide induces fungal resistance in postharvest Kyoho grapes by activating the salicylic acid-dependent pathway and inhibiting browning.

    PubMed

    Sun, Fei; Zhang, Pengying; Guo, Moran; Yu, Wenqian; Chen, Kaoshan

    2013-05-01

    Burdock fructooligosaccharide (BFO) is a natural elicitor from Arcitum lappa. The effects of BFO in controlling postharvest disease in grape, apple, banana, kiwi, citrus, strawberry, and pear were investigated. The disease index, decay percentage, and area under the disease progress curve indicated that BFO has general control effects on postharvest disease of fruits. Kyoho grapes were studied to elucidate the mechanism of BFO in boosting the resistance of grapes to Botrytis cinerea infection. BFO treatment induced upregulation of the npr1, pr1, pal, and sts genes, and inhibited the total phenol content decrease, which activated chitinase and β-1,3-glucanase. These results indicated that the salicylic acid-dependent signalling pathway was induced. The delayed colour change and peroxidase and polyphenoloxidase activity suggested that BFO delayed grape browning. The reduced respiration rate, weight loss, and titratable acidity prolonged the shelf life of postharvest grapes. BFO is a promising elicitor in postharvest disease control. PMID:23265522

  10. Burdock fructooligosaccharide induces fungal resistance in postharvest Kyoho grapes by activating the salicylic acid-dependent pathway and inhibiting browning.

    PubMed

    Sun, Fei; Zhang, Pengying; Guo, Moran; Yu, Wenqian; Chen, Kaoshan

    2013-05-01

    Burdock fructooligosaccharide (BFO) is a natural elicitor from Arcitum lappa. The effects of BFO in controlling postharvest disease in grape, apple, banana, kiwi, citrus, strawberry, and pear were investigated. The disease index, decay percentage, and area under the disease progress curve indicated that BFO has general control effects on postharvest disease of fruits. Kyoho grapes were studied to elucidate the mechanism of BFO in boosting the resistance of grapes to Botrytis cinerea infection. BFO treatment induced upregulation of the npr1, pr1, pal, and sts genes, and inhibited the total phenol content decrease, which activated chitinase and β-1,3-glucanase. These results indicated that the salicylic acid-dependent signalling pathway was induced. The delayed colour change and peroxidase and polyphenoloxidase activity suggested that BFO delayed grape browning. The reduced respiration rate, weight loss, and titratable acidity prolonged the shelf life of postharvest grapes. BFO is a promising elicitor in postharvest disease control.

  11. Changes in enzymatic activities and microbial properties in vermicompost of water hyacinth as affected by pre-composting and fungal inoculation: a comparative study of ergosterol and chitin for estimating fungal biomass.

    PubMed

    Pramanik, P

    2010-01-01

    In this experiment, three different fungal species, viz. Trichoderma viridae, Aspergillus niger and Phanerochaete chrysosporium, were inoculated in 7 day and 15 day partially decomposed water hyacinth to study their effect on enzymatic activities, microbial respiration and fungal biomass of the final stabilized product. The results suggested that increasing the duration of pre-composting from 7 days to 15 days did not show any significant effect on the activities of hydrolytic enzymes. Inoculation of fungi significantly (P < or = 0.05) increased cellulase, protease and acid and alkaline phosphatase activities. The highest value of ergosterol was recorded in A. niger-inoculated vermicomposts. Inoculation of P. chrysosporium in initial organic waste registered the highest chitin content in vermicompost. A comparison of fungal biomass and chitin content revealed a conversion factor of 2.628 with a standard deviation of 0.318. Due to significant correlation (r = 0.864), this conversion factor allows for the calculation of fungal biomass from chitin, which is comparatively more stable than ergosterol.

  12. Inhibitors of the hepatitis C virus NS3 protease with basic amine functionality at the P3-amino acid N-terminus: discovery and optimization of a new series of P2-P4 macrocycles.

    PubMed

    Harper, Steven; Ferrara, Marco; Crescenzi, Benedetta; Pompei, Marco; Palumbi, Maria Cecilia; DiMuzio, Jillian M; Donghi, Monica; Fiore, Fabrizio; Koch, Uwe; Liverton, Nigel J; Pesci, Silvia; Petrocchi, Alessia; Rowley, Michael; Summa, Vincenzo; Gardelli, Cristina

    2009-08-13

    In a follow-up to our recent disclosure of P2-P4 macrocyclic inhibitors of the hepatitis C virus (HCV) NS3 protease (e.g., 1, Chart 1), we report a new but related compound series featuring a basic amine at the N-terminus of the P3-amino acid residue. Replacement of the electroneutral P3-amino acid capping group (which is a feature of almost all tripeptide-like inhibitors of NS3 reported to date) with a basic group is not only tolerated but can result in advantageous cell based potency. Optimization of this new class of P3-amine based inhibitors gave compounds such as 25 and 26 that combine excellent cell based activity with pharmacokinetic properties that are attractive for an antiviral targeting HCV.

  13. Structure elucidation and inhibitory effects of self-assembled organotin(IV) esters of p-tolyl acetic acid on bacterial, fungal, brine shrimp, and potato tumor cells.

    PubMed

    Hussain, Mukhtiar; Ahmad, Muhammad Sheeraz; Hanif, Muhammad; Ali, Saqib; Mirza, Bushra

    2010-04-01

    Biologically active new organotin(IV) acetates (1-6) of p-tolyl acetic acid have been synthesized and characterized by multinuclear nuclear magnetic resonance ((1)H, (13)C) and mass spectrometry. The X-ray crystallographic data of compounds (3 and 4) were also collected. These studies show that in diorganotin(IV) compounds (compounds 1, 2, 3, and 6), tin exists in skew trapezoidal geometry with four strong and two weaker bonds, whereas triorganotin(IV) carboxylates (compounds 4 and 5) have four and five coordinated geometries in solution and solid form, respectively. These compounds were screened for a range of biological activities. Antibacterial activities were determined against six pathogenic bacterial strains, three Gram-positive and three Gram-negative; the activities were measured in terms of inhibition zones (mm). Antifungal activities were determined against six pathogenic fungal strains, cytotoxicity by the brine shrimp lethality assay, and antitumor activities by the crown gall tumor inhibition (potato disc) assay. PMID:20307145

  14. Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.

    PubMed

    Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

    2013-01-01

    During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors.

  15. Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs

    PubMed Central

    Hauser, Philippe M.; Burdet, Frédéric X.; Cissé, Ousmane H.; Keller, Laurent; Taffé, Patrick; Sanglard, Dominique; Pagni, Marco

    2010-01-01

    Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8’085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4’974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the “amino acid metabolism” category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans. PMID

  16. [Search for nucleic acid influencing, as well as membrane active, potential cancerostatic fungal metabolites using microbiological and cytological screening methods].

    PubMed

    Dornberger, K; Gutsche, W; Horschak, R; Zureck, A

    1978-01-01

    A prescreening program including microbiological and cytological assays was employed in search of potential cancerostatic antibiotics in crude extracts of mushrooms. The microbiological tests based on agar diffusion techniques consist of prophage induction test and BIP-test. All active compounds selected by these microbiological models are potential inhibitors of nucleic acid metabolism. Cytological assays on leukemia L 1210 cells have been carried out by microscopic examination and by evaluation using an electronic particle counter. Activity was expressed as decrease of the number of single cells caused by agglutination or lysis of cells, changes in cell surface area, dye exclusion, and increase of cell volume. A wide variety of mushrooms was demonstrated to exhibit interesting activities in some of these screening systems. The influence of primary metabolic products of mushrooms on microbiological models was studied additionally. In vivo assays have not yet been accomplished.

  17. Optimization of protease production by endophytic fungus, Alternaria alternata, isolated from an Australian native plant.

    PubMed

    Zaferanloo, Bita; Quang, Trung D; Daumoo, Smita; Ghorbani, Mahmood M; Mahon, Peter J; Palombo, Enzo A

    2014-06-01

    Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3-9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.

  18. 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

    PubMed Central

    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. PMID:26494731

  19. 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.

    PubMed

    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.

  20. Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

    PubMed

    Fernández-Marín, Hermógenes; Nash, David R; Higginbotham, Sarah; Estrada, Catalina; van Zweden, Jelle S; d'Ettorre, Patrizia; Wcislo, William T; Boomsma, Jacobus J

    2015-05-22

    Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens. PMID:25925100

  1. Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

    PubMed Central

    Fernández-Marín, Hermógenes; Nash, David R.; Higginbotham, Sarah; Estrada, Catalina; van Zweden, Jelle S.; d'Ettorre, Patrizia; Wcislo, William T.; Boomsma, Jacobus J.

    2015-01-01

    Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens. PMID:25925100

  2. Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

    PubMed

    Fernández-Marín, Hermógenes; Nash, David R; Higginbotham, Sarah; Estrada, Catalina; van Zweden, Jelle S; d'Ettorre, Patrizia; Wcislo, William T; Boomsma, Jacobus J

    2015-05-22

    Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

  3. Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease.

    PubMed

    Chu, Yuan; Zhou, Fan; Liu, Yang; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae. PMID:25818483

  4. Identification of potential mitochondrial CLPXP protease interactors and substrates suggests its central role in energy metabolism

    PubMed Central

    Fischer, Fabian; Langer, Julian D.; Osiewacz, Heinz D.

    2015-01-01

    Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP’s substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes. PMID:26679294

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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. Oxygenation by COX-2 (cyclo-oxygenase-2) of 3-HETE (3-hydroxyeicosatetraenoic acid), a fungal mimetic of arachidonic acid, produces a cascade of novel bioactive 3-hydroxyeicosanoids

    PubMed Central

    2005-01-01

    Cyclo-oxygenases-1/2 (COX-1/2) catalyse the oxygenation of AA (arachidonic acid) and related polyunsaturated fatty acids to endoperoxide precursors of prostanoids. COX-1 is referred to as a constitutive enzyme involved in haemostasis, whereas COX-2 is an inducible enzyme expressed in inflammatory diseases and cancer. The fungus Dipodascopsis uninucleata has been shown by us to convert exogenous AA into 3(R)-HETE [3(R)-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid]. 3R-HETE is stereochemically identical with AA, except that a hydroxy group is attached at its C-3 position. Molecular modelling studies with 3-HETE and COX-1/2 revealed a similar enzyme–substrate structure as reported for AA and COX-1/2. Here, we report that 3-HETE is an appropriate substrate for COX-1 and -2, albeit with a lower activity of oxygenation than AA. Oxygenation of 3-HETE by COX-2 produced a novel cascade of 3-hydroxyeicosanoids, as identified with EI (electron impact)–GC–MS, LC–MS–ES (electrospray) and LC–MS–API (atmospheric pressure ionization) methods. Evidence for in vitro production of 3-hydroxy-PGE2 (3-hydroxy-prostaglandin E2) was obtained upon infection of HeLa cells with Candida albicans at an MOI (multiplicity of infection) of 100. Analogous to interaction of AA and aspirin-treated COX-2, 3-HETE was transformed by acetylated COX-2 to 3,15-di-HETE (3,15-dihydroxy-HETE), whereby C-15 showed the (R)-stereochemistry. 3-Hydroxy-PGs are potent biologically active compounds. Thus 3-hydroxy-PGE2 induced interleukin-6 gene expression via the EP3 receptor (PGE2 receptor 3) in A549 cells, and raised cAMP levels via the EP4 receptor in Jurkat cells. Moreover, 3R,15S-di-HETE triggered the opening of the K+ channel in HTM (human trabecular meshwork) cells, as measured by the patch–clamp technique. Since many fatty acid disorders are associated with an ‘escape’ of 3-hydroxy fatty acids from the β-oxidation cycle, the production of 3-hydroxyeicosanoids may be critical in

  8. Cloning and identification of Fv-cmp, a protease from Fusarium verticillioides that truncates Zea mays and Arabidopsis thaliana class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, that were originally identified as proteins that truncate class IV chitinases of maize during ear rot. Cmps from Bipolaris zeicola and Stenocarpella maydis have been characterized, but the identities of the proteases h...

  9. Fungal Skin Infections

    MedlinePlus

    ... Fungal Skin Infections Overview of Fungal Skin Infections Candidiasis Overview of Dermatophytoses (Ringworm, Tinea) Athlete's Foot Jock ... are caused by yeasts (such as Candida —see Candidiasis ) or dermatophytes, such as Epidermophyton, Microsporum, and Trichophyton ( ...

  10. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    PubMed Central

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases. Images PMID:3186696

  11. The Lon AAA+ protease.

    PubMed

    Gur, Eyal

    2013-01-01

    As the first ATP-dependent protease to be identified, Lon holds a special place in the history of cellular biology. In fact, the concept of ATP-dependent protein degradation was established through the findings that led to the discovery of Lon. Therefore, this chapter begins with a historical perspective, describing the milestones that led to the discovery of Lon and ATP-dependent proteolysis, starting from the early findings in the 1960s until the demonstration of Lon's ATP-dependent proteolytic activity in vitro, in 1981. Most of our knowledge on Lon derives from studies of the Escherichia coli Lon ortholog, and, therefore, most of this chapter relates to this particular enzyme. Nonetheless, Lon is not only found in most bacterial species, it is also found in Archaea and in the mitochondrion and chloroplast of eukaryotic cells. Therefore many of the conclusions gained from studies on the E. coli enzyme are relevant to Lon proteases in other organisms. Lon, more than any other bacterial or organellar protease, is associated with the degradation of misfolded proteins and protein quality control. In addition, Lon also degrades many regulatory proteins that are natively folded, thus it also plays a prominent role in regulation of physiological processes. Throughout the years, many Lon substrates have been identified, confirming its role in the regulation of diverse cellular processes, including cell division, DNA replication, differentiation, and adaptation to stress conditions. Some examples of these functions are described and discussed here, as is the role of Lon in the degradation of misfolded proteins and in protein quality control. Finally, this chapter deals with the exquisite sensitivity of protein degradation inside a cell. How can a protease distinguish so many substrates from cellular proteins that should not be degraded? Can the specificity of a protease be regulated according to the physiological needs of a cell? This chapter thus broadly discusses the

  12. Laundry performance of subtilisin proteases.

    PubMed

    Wolff, A M; Showell, M S; Venegas, M G; Barnett, B L; Wertz, W C

    1996-01-01

    Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.

  13. Fungal stealth technology.

    PubMed

    Rappleye, Chad A; Goldman, William E

    2008-01-01

    Medically important fungi range from commensal organisms that cause opportunistic infections to primary fungal pathogens that can cause disease in immunocompetent hosts. Host phagocyte-expressed pattern-recognition receptors represent one obstacle to infection, and the extent to which fungal cells can evade detection by host receptors helps shape their pathogenic potential. This review highlights recently defined mechanisms employed by successful fungal pathogens to conceal their immunostimulatory molecular signatures from leukocyte receptors or to disrupt host response signals. Continued improvements in our understanding of these fungal stealth mechanisms should provide new options for future therapeutics to expose these fungal pathogens and limit their virulence capacity.

  14. Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis

    PubMed Central

    Desjardins, Christopher A.; Champion, Mia D.; Holder, Jason W.; Muszewska, Anna; Goldberg, Jonathan; Bailão, Alexandre M.; Brigido, Marcelo Macedo; Ferreira, Márcia Eliana da Silva; Garcia, Ana Maria; Grynberg, Marcin; Gujja, Sharvari; Heiman, David I.; Henn, Matthew R.; Kodira, Chinnappa D.; León-Narváez, Henry; Longo, Larissa V. G.; Ma, Li-Jun; Malavazi, Iran; Matsuo, Alisson L.; Morais, Flavia V.; Pereira, Maristela; Rodríguez-Brito, Sabrina; Sakthikumar, Sharadha; Salem-Izacc, Silvia M.; Sykes, Sean M.; Teixeira, Marcus Melo; Vallejo, Milene C.; Walter, Maria Emília Machado Telles; Yandava, Chandri; Young, Sarah; Zeng, Qiandong; Zucker, Jeremy; Felipe, Maria Sueli; Goldman, Gustavo H.; Haas, Brian J.; McEwen, Juan G.; Nino-Vega, Gustavo; Puccia, Rosana; San-Blas, Gioconda; Soares, Celia Maria de Almeida; Birren, Bruce W.; Cuomo, Christina A.

    2011-01-01

    Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of

  15. Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

    PubMed

    Desjardins, Christopher A; Champion, Mia D; Holder, Jason W; Muszewska, Anna; Goldberg, Jonathan; Bailão, Alexandre M; Brigido, Marcelo Macedo; Ferreira, Márcia Eliana da Silva; Garcia, Ana Maria; Grynberg, Marcin; Gujja, Sharvari; Heiman, David I; Henn, Matthew R; Kodira, Chinnappa D; León-Narváez, Henry; Longo, Larissa V G; Ma, Li-Jun; Malavazi, Iran; Matsuo, Alisson L; Morais, Flavia V; Pereira, Maristela; Rodríguez-Brito, Sabrina; Sakthikumar, Sharadha; Salem-Izacc, Silvia M; Sykes, Sean M; Teixeira, Marcus Melo; Vallejo, Milene C; Walter, Maria Emília Machado Telles; Yandava, Chandri; Young, Sarah; Zeng, Qiandong; Zucker, Jeremy; Felipe, Maria Sueli; Goldman, Gustavo H; Haas, Brian J; McEwen, Juan G; Nino-Vega, Gustavo; Puccia, Rosana; San-Blas, Gioconda; Soares, Celia Maria de Almeida; Birren, Bruce W; Cuomo, Christina A

    2011-10-01

    Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of

  16. Purification and characterization of a serine protease from Cucumis trigonus Roxburghi.

    PubMed

    Asif-Ullah, Mufti; Kim, Key-Sun; Yu, Yeon Gyu

    2006-05-01

    Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family. PMID:16603211

  17. From proteases to proteomics.

    PubMed

    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).

  18. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    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). PMID:11274481

  19. Peptidotriazoles with antimicrobial activity against bacterial and fungal plant pathogens.

    PubMed

    Güell, Imma; Micaló, Lluís; Cano, Laura; Badosa, Esther; Ferre, Rafael; Montesinos, Emilio; Bardají, Eduard; Feliu, Lidia; Planas, Marta

    2012-01-01

    We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH(2)) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6-12.5 μM), and against the fungi Fusarium oxysporum (MIC<6.2-12.5 μM) with low hemolytic activity (0-23% at 50 μM), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents. PMID:22198367

  20. Fungal genome sequencing: basic biology to biotechnology.

    PubMed

    Sharma, Krishna Kant

    2016-08-01

    The genome sequences provide a first glimpse into the genomic basis of the biological diversity of filamentous fungi and yeast. The genome sequence of the budding yeast, Saccharomyces cerevisiae, with a small genome size, unicellular growth, and rich history of genetic and molecular analyses was a milestone of early genomics in the 1990s. The subsequent completion of fission yeast, Schizosaccharomyces pombe and genetic model, Neurospora crassa initiated a revolution in the genomics of the fungal kingdom. In due course of time, a substantial number of fungal genomes have been sequenced and publicly released, representing the widest sampling of genomes from any eukaryotic kingdom. An ambitious genome-sequencing program provides a wealth of data on metabolic diversity within the fungal kingdom, thereby enhancing research into medical science, agriculture science, ecology, bioremediation, bioenergy, and the biotechnology industry. Fungal genomics have higher potential to positively affect human health, environmental health, and the planet's stored energy. With a significant increase in sequenced fungal genomes, the known diversity of genes encoding organic acids, antibiotics, enzymes, and their pathways has increased exponentially. Currently, over a hundred fungal genome sequences are publicly available; however, no inclusive review has been published. This review is an initiative to address the significance of the fungal genome-sequencing program and provides the road map for basic and applied research.

  1. The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense.

    PubMed

    van Esse, H Peter; Van't Klooster, John W; Bolton, Melvin D; Yadeta, Koste A; van Baarlen, Peter; Boeren, Sjef; Vervoort, Jacques; de Wit, Pierre J G M; Thomma, Bart P H J

    2008-07-01

    Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum). During growth in the apoplast, the fungus establishes disease by secreting effector proteins, 10 of which have been characterized. We have previously shown that the Avr2 effector interacts with the apoplastic tomato Cys protease Rcr3, which is required for Cf-2-mediated immunity. We now show that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis thaliana causes enhanced susceptibility toward extracellular fungal pathogens, including Botrytis cinerea and Verticillium dahliae, and microarray analysis showed that Avr2 expression triggers a global transcriptome reflecting pathogen challenge. Cys protease activity profiling showed that Avr2 inhibits multiple extracellular Arabidopsis Cys proteases. In tomato, Avr2 expression caused enhanced susceptibility toward Avr2-defective C. fulvum strains and also toward B. cinerea and V. dahliae. Cys protease activity profiling in tomato revealed that, in this plant also, Avr2 inhibits multiple extracellular Cys proteases, including Rcr3 and its close relative Pip1. Finally, silencing of Avr2 significantly compromised C. fulvum virulence on tomato. We conclude that Avr2 is a genuine virulence factor of C. fulvum that inhibits several Cys proteases required for plant basal defense.

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

    PubMed

    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.

  3. Coagulation factor XII protease domain crystal structure

    PubMed Central

    Pathak, M; Wilmann, P; Awford, J; Li, C; Hamad, BK; Fischer, PM; Dreveny, I; Dekker, LV; Emsley, J

    2015-01-01

    Background Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI. Objective To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain. Methods and results A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates. Conclusions These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation. PMID:25604127

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

    PubMed

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

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

  5. Protease signalling: the cutting edge

    PubMed Central

    Turk, Boris; Turk, Dus̆an; Turk, Vito

    2012-01-01

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. PMID:22367392

  6. Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Sutherland, Katherine; Parry, Chris M.; Cane, Patricia A.; Coovadia, Ashraf; Kuhn, Louise; Hunt, Gillian

    2016-01-01

    Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of

  7. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    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.

  8. graal: a Drosophila gene coding for several mosaic serine proteases.

    PubMed

    Munier, Anne Isabelle; Medzhitov, Ruslan; Janeway, Charles A; Doucet, Daniel; Capovilla, Maria; Lagueux, Marie

    2004-10-01

    Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge.

  9. The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

    PubMed

    Pöll, Verena; Denk, Ursula; Shen, Horng-Der; Panzani, Raphael C; Dissertori, Oliver; Lackner, Peter; Hemmer, Wolfgang; Mari, Adriano; Crameri, Reto; Lottspeich, Friedrich; Rid, Raphaela; Richter, Klaus; Breitenbach, Michael; Simon-Nobbe, Birgit

    2009-04-01

    Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine

  10. Plant hormones: a fungal point of view.

    PubMed

    Chanclud, Emilie; Morel, Jean-Benoit

    2016-10-01

    Most classical plant hormones are also produced by pathogenic and symbiotic fungi. The way in which these molecules favour the invasion of plant tissues and the development of fungi inside plant tissues is still largely unknown. In this review, we examine the different roles of such hormone production by pathogenic fungi. Converging evidence suggests that these fungal-derived molecules have potentially two modes of action: (i) they may perturb plant processes, either positively or negatively, to favour invasion and nutrient uptake; and (ii) they may also act as signals for the fungi themselves to engage appropriate developmental and physiological processes adapted to their environment. Indirect evidence suggests that abscisic acid, gibberellic acid and ethylene produced by fungi participate in pathogenicity. There is now evidence that auxin and cytokinins could be positive regulators required for virulence. Further research should establish whether or not fungal-derived hormones act like other fungal effectors.

  11. Kinetics of alkaline protease production by Streptomyces griseoflavus PTCC1130

    PubMed Central

    Hosseini, Seyed Vesal; Saffari, Zahra; Farhanghi, Ali; Atyabi, Seyed Mohammad; Norouzian, Dariush

    2016-01-01

    Background and Objectives: Proteases are a group of enzymes that catalyze the degradation of proteins resulting in the production of their amino acid constituents. They are the most important group of industrial enzymes which account for about 60% of total enzymes in the market and produced mainly by microorganisms. The attempts were made to study the kinetic parameters of protease produced by Streptomyces griseoflavus PTCC1130. Materials and Methods: Streptomyces griseoflavus PTCC1130 was grown on casein agar. Different media such as BM1, BM2, BM3 and BM4 were prepared. Data obtained from growth and protease production were subjected to kinetics evaluation. Casein was used as substrate for protease activity and the released soluble peptide bearing aromatic amino acid were quantified by Folin Cioclateaue reagent. Protein content of the enzyme and the sugar utilized by the organism were estimated by Bradford and Miller’s methods respectively. Results: Basal Medium named as BM1, BM2, BM3 and BM4(50 mL in 250 mL Erlen Meyer flasks) were screened out to evaluate protease production by Streptomyces griseoflavus PTCC1130. They were inoculated with known amount of seed culture and kept on rotary shaker. To obtain the specific growth rate, wet weight of biomass was plotted against the time. The clarified supernatant was used for the analysis of protease by measuring the soluble peptide containing aromatic amino acid residues employing Folin Cioclateaue reagent. Our results showed that maximum level of enzyme production (14035 U/L) was occurred at late exponential phase using Basal Medium supplemented with zinc sulfate (0.5g/L), casein (10g/L) at pH 6.5. Conclusions: A kinetic study of protease production by Streptomyces griseoflavus PTCC1130 provided highly quantitative information regarding the behavior of a system, which is essential to study the fermentation process. Exploitation of such kinetics analysis would be useful in commercialization of microbial enzyme

  12. Protease degradable electrospun fibrous hydrogels

    PubMed Central

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

    2015-01-01

    Electrospun nanofibers 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 (MMPs). 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 fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications. PMID:25799370

  13. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    NASA Astrophysics Data System (ADS)

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-03-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

  14. A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

    PubMed

    Wan, Hu; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Lee, Kyung Yong; Jin, Byung Rae

    2014-01-01

    Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions.

  15. Protease addition to increase yield and fermentation rate in dry grind ethanol production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a small scale laboratory procedure (100g shake flasks) for ethanol production from corn, the effects of acid protease addition during the fermentation step were evaluated. The batch fermentations were conducted in duplicate using standard conditions and with protease addition during fermentati...

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

    PubMed

    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. PMID:26559682

  17. Fungal Genomics Program

    SciTech Connect

    Grigoriev, Igor

    2012-03-12

    The JGI Fungal Genomics Program aims to scale up sequencing and analysis of fungal genomes to explore the diversity of fungi important for energy and the environment, and to promote functional studies on a system level. Combining new sequencing technologies and comparative genomics tools, JGI is now leading the world in fungal genome sequencing and analysis. Over 120 sequenced fungal genomes with analytical tools are available via MycoCosm (www.jgi.doe.gov/fungi), a web-portal for fungal biologists. Our model of interacting with user communities, unique among other sequencing centers, helps organize these communities, improves genome annotation and analysis work, and facilitates new larger-scale genomic projects. This resulted in 20 high-profile papers published in 2011 alone and contributing to the Genomics Encyclopedia of Fungi, which targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts). Our next grand challenges include larger scale exploration of fungal diversity (1000 fungal genomes), developing molecular tools for DOE-relevant model organisms, and analysis of complex systems and metagenomes.

  18. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    SciTech Connect

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. |

    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.

  19. Fungal Vaccines and Immunotherapeutics

    PubMed Central

    Santos, Evelyn; Levitz, Stuart M.

    2014-01-01

    Concomitant with the increased prevalence of immunocompromised persons, invasive fungal infections have become considerably more frequent in the last 50 years. High mortality rates caused by invasive mycoses and high morbidity because of intractable mucosal infections have created an unmet need for innovative prophylactic and therapeutic strategies against fungal pathogens. Several immunotherapeutics and vaccines are in development to address this need, although one has yet to reach the clinic. This review focuses on past and current immunotherapeutic and vaccine strategies being tested to either prevent or treat fungal infections, as well as the challenges associated with their development. PMID:25368016

  20. Fungal cell-wall lytic enzymes, antifungal metabolite(s) production, and characterization from Streptomyces exfoliatus MT9 for controlling fruit-rotting fungi.

    PubMed

    Choudhary, Bharti; Nagpure, Anand; Gupta, Rajinder K

    2014-12-01

    An antifungal actinomycete strain MT9 was isolated from Loktak Lake, Manipur, India and its cultural characteristics, fatty acid methyl ester, 16S rRNA gene analysis suggests that strain MT9 is identical to Streptomyces exfoliatus. Strain MT9 displayed strong and broad-spectrum antagonism towards several fruit-rotting fungi by mycelial growth suppression. Crude fungal cell-wall lytic enzymes, i.e., chitinase, β-1,3-glucanase, and protease produced by S. exfoliatus MT9 were optimally active at pH 8.0 and 50 °C, pH 5.0 and 60 °C, pH 9.0 and 70 °C, respectively. All three mycolytic enzymes had good stability over a wide pH range of 5.0-10.0, with protease being more thermostable than both chitinase and β-1,3-glucanase. Interestingly zymogram analysis revealed that S. exfoliatus MT9 secretes six distinct chitinase isoenzymes with approximate molecular weights of 9.42, 13.93, 27.87, 36.43, 54.95, 103.27 kDa, six active protease isoenzymes with apparent molecular weights of 12.45, 30.20, 37.45, 46.32, 52.46, 131.46 kDa, and an active band of 119.39 kDa as β-1,3-glucanase enzyme. Extracellular fluid and its organic solvent extracts also exhibited inhibitory activity to various fruit-rotting fungi. The MIC value of n-butanol extract was 2-25 µg/ml against tested fruit-rotting fungi. Antifungal secondary metabolite(s) was found to be polyene in nature. To the best of our knowledge, this is the first report on extracellular production of fungal cell-wall lytic enzymes and antifungal metabolites by bioactive S. exfoliatus MT9 under submerged fermentation.

  1. Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste.

    PubMed

    Anandharaj, Marimuthu; Sivasankari, Balayogan; Siddharthan, Nagarajan; Rani, Rizwana Parveen; Sivakumar, Subramaniyan

    2016-04-01

    Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40 °C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60 °C and pH 10. Ca(2+), Mn(2+), and Mg(2+) ions activated the protease, while Hg(2+), Cu(2+), Zn(2+), and Fe(2+) greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca(2+), which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications. PMID:26749296

  2. JGI Fungal Genomics Program

    SciTech Connect

    Grigoriev, Igor V.

    2011-03-14

    Genomes of energy and environment fungi are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). Its key project, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts), and explores fungal diversity by means of genome sequencing and analysis. Over 50 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functional genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such 'parts' suggested by comparative genomics and functional analysis in these areas are presented here

  3. Fungal Biofilm Resistance

    PubMed Central

    Ramage, Gordon; Rajendran, Ranjith; Sherry, Leighann; Williams, Craig

    2012-01-01

    Fungal biofilm infections have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. This paper aims to illustrate the importance of fungal biofilms, particularly Candida albicans, and discusses some of the key fungal biofilm resistance mechanisms that include, extracellular matrix (ECM), efflux pump activity, persisters, cell density, overexpression of drug targets, stress responses, and the general physiology of the cell. The paper demonstrates the multifaceted nature of fungal biofilm resistance, which encompasses some of the newest data and ideas in the field. PMID:22518145

  4. Systemic opportunistic fungal infections.

    PubMed Central

    Vanbreuseghem, R.; Vroey, C. D.

    1979-01-01

    The clinical manifestations of "opportunistic" fungal infections in compromised hosts, asthenomycoses, differ from those caused by the same fungus in otherwise normal people. Examples are given on the field of dermatophytoses, aspergillosis, candidiasis and cryptococcosis. PMID:523345

  5. Fungal Nail Infection (Onychomycosis)

    MedlinePlus

    ... vinegar, vitamin E oil, Vicks® VapoRub®, or tea tree oil. When to Seek Medical Care Fungal nail ... Trusted Links Related diseases: Psoriasis View all diseases Community: Discussion Forum Skinmatters Blog About Us | Terms of ...

  6. Fungal Eye Infections

    MedlinePlus

    ... Zoonotic Infectious Disease Division of Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch Fungal Eye Infections Recommend on ... Zoonotic Infectious Disease Division of Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch File Formats Help: How do ...

  7. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  8. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles.

  9. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  10. Fungal diagnostics in pneumonia.

    PubMed

    Lease, Erika D; Alexander, Barbara D

    2011-12-01

    Fungal pneumonia is increasingly common, particularly in highly immunosuppressed patients, such as solid organ or hematopoietic stem cell transplant recipients, and the diagnosis is evolving. Although standard techniques such as microscopy and culture remain the mainstays of diagnosis, relatively recent advances in serological and molecular testing are important additions to the field. This article reviews the laboratory tools used to diagnose fungal respiratory disease. PMID:22167394

  11. Fungal Diagnostics in Pneumonia

    PubMed Central

    Lease, Erika D.; Alexander, Barbara D.

    2014-01-01

    Fungal pneumonia is increasingly common, particularly in highly immunosuppressed patients, such as solid organ or hematopoietic stem cell transplant recipients, and the diagnosis is evolving. While standard techniques such as microscopy and culture remain the mainstay of diagnosis, relatively recent advances in serologic and molecular testing are important additions to the field. This chapter will review the laboratory tools used to diagnose fungal respiratory disease. PMID:22167394

  12. Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in β-oxidation of fatty acids

    PubMed Central

    Kurochkin, Igor V; Mizuno, Yumi; Konagaya, Akihiko; Sakaki, Yoshiyuki; Schönbach, Christian; Okazaki, Yasushi

    2007-01-01

    Peroxisomes play an important role in β-oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post-translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C-terminal PTS1 and the N-terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N-terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal β-oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N-terminal fragment. Tysnd1 expression is induced by the proliferator-activated receptor α agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1-mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of β-oxidation. PMID:17255948

  13. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  14. [Pathogenesis of invasive fungal infections].

    PubMed

    Garcia-Vidal, Carolina; Carratalà, Jordi

    2012-03-01

    Invasive fungal infections remain a life-threatening disease. The development of invasive fungal disease is dependent on multiple factors, such us colonization and efficient host immune response. We aimed to review the pathogenesis of invasive fungal infections, in particular, those caused by Candida and Aspergillus. For this we propose, to describe the fungal characteristics, to detail the host defence mechanisms against fungus and to analyse the host risk factors for invasive fungal infection.

  15. A new organic solvent tolerant protease from Bacillus pumilus 115b.

    PubMed

    Rahman, Raja Noor Zaliha Raja Abd; Mahamad, Shalihah; Salleh, Abu Bakar; Basri, Mahiran

    2007-07-01

    Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.

  16. Serine proteases of parasitic helminths.

    PubMed

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  17. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  18. The evolution of fungal metabolic pathways.

    PubMed

    Wisecaver, Jennifer H; Slot, Jason C; Rokas, Antonis

    2014-12-01

    Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters

  19. Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

    PubMed Central

    Gilbert, J V; Plaut, A G; Wright, A

    1991-01-01

    The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention. Images PMID:1987065

  20. Comparative genomics of mycobacterial proteases.

    PubMed

    Ribeiro-Guimarães, Michelle Lopes; Pessolani, Maria Cristina Vidal

    2007-01-01

    Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.

  1. Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

    PubMed Central

    Salganik, Maxim; Venkatakrishnan, Balasubramanian; Bennett, Antonette; Lins, Bridget; Yarbrough, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection. PMID:22915820

  2. The Cladosporium fulvum Virulence Protein Avr2 Inhibits Host Proteases Required for Basal Defense[W][OA

    PubMed Central

    van Esse, H. Peter; van't Klooster, John W.; Bolton, Melvin D.; Yadeta, Koste A.; van Baarlen, Peter; Boeren, Sjef; Vervoort, Jacques; de Wit, Pierre J.G.M.; Thomma, Bart P.H.J.

    2008-01-01

    Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum). During growth in the apoplast, the fungus establishes disease by secreting effector proteins, 10 of which have been characterized. We have previously shown that the Avr2 effector interacts with the apoplastic tomato Cys protease Rcr3, which is required for Cf-2–mediated immunity. We now show that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis thaliana causes enhanced susceptibility toward extracellular fungal pathogens, including Botrytis cinerea and Verticillium dahliae, and microarray analysis showed that Avr2 expression triggers a global transcriptome reflecting pathogen challenge. Cys protease activity profiling showed that Avr2 inhibits multiple extracellular Arabidopsis Cys proteases. In tomato, Avr2 expression caused enhanced susceptibility toward Avr2-defective C. fulvum strains and also toward B. cinerea and V. dahliae. Cys protease activity profiling in tomato revealed that, in this plant also, Avr2 inhibits multiple extracellular Cys proteases, including Rcr3 and its close relative Pip1. Finally, silencing of Avr2 significantly compromised C. fulvum virulence on tomato. We conclude that Avr2 is a genuine virulence factor of C. fulvum that inhibits several Cys proteases required for plant basal defense. PMID:18660430

  3. Fungal endocarditis: current challenges.

    PubMed

    Tattevin, Pierre; Revest, Matthieu; Lefort, Agnès; Michelet, Christian; Lortholary, Olivier

    2014-10-01

    Whilst it used to affect mostly intravenous drug users and patients who underwent valvular surgery with suboptimal infection control procedures, fungal endocarditis is now mostly observed in patients with severe immunodeficiency (onco-haematology), in association with chronic central venous access and broad-spectrum antibiotic use. The incidence of fungal endocarditis has probably decreased in most developed countries with access to harm-reduction policies (i.e. needle exchange programmes) and with improved infection control procedures during cardiac surgery. Use of specific blood culture bottles for diagnosis of fungal endocarditis has decreased due to optimisation of media and automated culture systems. Meanwhile, the advent of rapid techniques, including fungal antigen detection (galactomannan, mannan/anti-mannan antibodies and β-1,3-d-glucans) and PCR (e.g. universal fungal PCR targeting 18S rRNA genes), shall improve sensitivity and reduce diagnostics delays, although limited data are available on their use for the diagnosis of fungal endocarditis. New antifungal agents available since the early 2000s may represent dramatic improvement for fungal endocarditis: (i) a new class, the echinocandins, has the potential to improve the management of Candida endocarditis owing to its fungicidal effect on yeasts as well as tolerability of increased dosages; and (ii) improved survival in patients with invasive aspergillosis with voriconazole compared with amphotericin B, and this may apply to Aspergillus sp. endocarditis as well, although its prognosis remains dismal. These achievements may allow selected patients to be cured with prolonged medical treatment alone when surgery is considered too risky.

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

    NASA Astrophysics Data System (ADS)

    Jin, Xin; Li, Ju-Fang; Huang, Ping-Ying; Dong, Xu-Yan; Guo, Lu-Lu; Yang, Liang; Cao, Yuan-Cheng; Wei, Fang; Zhao, Yuan-Di; Chen, Hong

    2010-07-01

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

  5. Alkaline protease from Neurospora crassa. Purification and partial characterization

    SciTech Connect

    Lindberg, R.A.; Eirich, L.D.; Price, J.S.; Wolfinbarger, L. Jr.; Drucker, H.

    1981-01-25

    A simple purification procedure was developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: (1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; (2) heat treatment, during which the inducer is digested by the protease; and (3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. The preparation was homogeneous and had a molecular weight of approx. 30,500. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45/sup 0/C, and susceptible to autodigestion. Hydrolysis of the ..beta.. chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.

  6. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  7. Plant collagenase: unique collagenolytic activity of cysteine proteases from ginger.

    PubMed

    Kim, Misook; Hamilton, Susan E; Guddat, Luke W; Overall, Christopher M

    2007-12-01

    Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516-1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate. PMID:17920199

  8. Protease activity in protein-free NS0 myeloma cell cultures.

    PubMed

    Spens, Erika; Häggström, Lena

    2005-01-01

    Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.

  9. Allergic Fungal Rhinosinusitis.

    PubMed

    Hoyt, Alice E W; Borish, Larry; Gurrola, José; Payne, Spencer

    2016-01-01

    This article reviews the history of allergic fungal rhinosinusitis and the clinical, pathologic, and radiographic criteria necessary to establish its diagnosis and differentiate this disease from other types of chronic rhinosinusitis. Allergic fungal rhinosinusitis is a noninvasive fungal form of sinus inflammation characterized by an often times unilateral, expansile process in which the typical allergic "peanut-butter-like" mucin contributes to the formation of nasal polyps, hyposmia/anosmia, and structural changes of the face. IgE sensitization to fungi is a necessary, but not sufficient, pathophysiologic component of the disease process that is also defined by microscopic visualization of mucin-containing fungus and characteristic radiological imaging. This article expounds on these details and others including the key clinical and scientific distinctions of this diagnosis, the pathophysiologic mechanisms beyond IgE-mediated hypersensitivity that must be at play, and areas of current and future research. PMID:27393774

  10. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  11. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  12. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  13. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination1

    PubMed Central

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C.; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A.L.

    2015-01-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. PMID:26048883

  14. Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Coovadia, Ashraf; Kuhn, Louise; Abrams, Elaine J.; Strehlau, Renate; Morris, Lynn

    2015-01-01

    Abstract Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection. PMID:25919760

  15. Long-term fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage.

    PubMed

    Coda, Rossana; Rizzello, Carlo G; Nigro, Franco; De Angelis, Maria; Arnault, Philip; Gobbetti, Marco

    2008-12-01

    The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.

  16. Polyethylene glycol (PEG) gel arrays for differentiating oligopeptide fragments and on-chip protease assays.

    PubMed

    Zhu, Qingdi; Yang, Kun-Lin

    2016-03-15

    Polyethylene glycol (PEG) hydrogel is permeable to biomolecules, but its permeability depends on the molecular weight of monomers and the concentration of monomer solutions. In this study, we show that PEG hydrogel made from 20% to 30% of PEG700 monomer is permeable to amino acids yet impermeable to oligopeptides. Because of its unique permeability, the gel can be used to detect protease by separating amino acids from oligopeptides when proteases cleave the oligopeptides and release amino acids. Based on this principle, an UV crosslinked gel array is fabricated on a chip for simultaneous detection of protease in up to 40 samples with only 1 µl of volume required for each sample. As a proof of concept, the on-chip protease assays are used to detect trypsin in buffer and serum. The detection limits are 1.2 nM in buffer and 17.7 nM in serum, which are comparable to conventional protease assays. Moreover, because only 1 µl of liquid is required, as little as 1.2 fmol of trypsin can be detected by using the on-chip assay. The protease assay also shows good specificity for trypsin and chymotrypsin. The gel array chip could be a useful miniaturized platform for high-throughput detection of different proteases and screening of their inhibitors. PMID:26569443

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

    PubMed

    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.

  18. Who Gets Fungal Infections?

    MedlinePlus

    ... infections can also happen in people without weak immune systems Fungal infections that are not life-threatening, such ... likely to cause an infection. People with weak immune systems Infections that happen because a person’s immune system ...

  19. Endotoxin, ergosterol, muramic acid and fungal DNA in dust from schools in Johor Bahru, Malaysia--Associations with rhinitis and sick building syndrome (SBS) in junior high school students.

    PubMed

    Norbäck, Dan; Hashim, Jamal Hisham; Markowicz, Pawel; Cai, Gui-Hong; Hashim, Zailina; Ali, Faridah; Larsson, Lennart

    2016-03-01

    This paper studied associations between ocular symptoms, rhinitis, throat and dermal symptoms, headache and fatigue in students by ethnicity and in relation to exposure to chemical microbial markers and fungal DNA in vacuumed dust in schools in Malaysia. A total of 462 students from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated (96% response rate). Dust was vacuumed from 32 classrooms and analysed for levels of five types of endotoxin as 3-hydroxy fatty acids (C10, C12, C14, C16 and C18 3-OH), muramic acid, ergosterol and five sequences of fungal DNA. Multiple logistic regression was applied. Totally 11.9% reported weekly ocular symptoms, 18.8% rhinitis, 15.6% throat and 11.1% dermal symptoms, 20.6% headache and 22.1% tiredness. Totally 21.1% reported pollen or furry pet allergy (atopy) and 22.0% parental asthma or allergy. Chinese students had less headache than Malay and Indian had less rhinitis and less tiredness than Malay. Parental asthma/allergy was a risk factor for ocular (odds ratio=3.79) and rhinitis symptoms (OR=3.48). Atopy was a risk factor for throat symptoms (OR=2.66), headache (OR=2.13) and tiredness (OR=2.02). There were positive associations between amount of fine dust in the dust samples and ocular symptoms (p<0.001) and rhinitis (p=0.006). There were positive associations between C14 3-OH and rhinitis (p<0.001) and between C18 3-OH and dermal symptoms (p=0.007). There were negative (protective) associations between levels of total endotoxin (LPS) (p=0.004) and levels of ergosterol (p=0.03) and rhinitis and between C12 3-OH and throat symptoms (p=0.004). In conclusion, the amount of fine dust in the classroom was associated with rhinitis and other SBS symptoms and improved cleaning of the schools is important. Endotoxin in the school dust seems to be mainly protective for rhinitis and throat symptoms but different types of endotoxin could have different effects. The ethnic differences in symptoms among the students

  20. Cloning, nucleotide sequence, and expression of Achromobacter protease I gene.

    PubMed

    Ohara, T; Makino, K; Shinagawa, H; Nakata, A; Norioka, S; Sakiyama, F

    1989-12-01

    Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini. PMID:2684982

  1. Overview of fungal rhinosinusitis.

    PubMed

    Chakrabarti, Arunaloke; Das, Ashim; Panda, Naresh K

    2004-10-01

    The incidence of fungal rhinosinusitis has increased to such extent in recent years that fungal infection should be considered in all patients with chronic rhino sinusitis. In India though the disease was reported earlier only from northern regions of this country, nowadays the disease is increasingly diagnosed from other parts as well. The disease has been categorized with possible five types: acute necrothing (fulminant), chronic invasive, chronic granulomatous invasive, fungal hall (sinus mycetoma), allergic. The first three types are tissue-invasive and the last two are non-invasive fungal rhinosinusitis. However, the categorization is still controversial and open to discussion. Chronic fungal rhinosinusitis can occur in otherwise healthy host and Aspergillus flavus is the common etiological agent in Indian scenario. The pathophys iologic mechanism of the disease remains unclear. It may represent an allergic IgE response, a cell-mediated reaction, or a combination of two. Early diagnosis may prevent multiple surgical procedures and lead to effective treatment. Histopathology and radio-imaging techniques help to distinguish different types and delineate extension of disease process. Culture helps to identify the responsible etiological agent. The presence or absence oj precipitating antibody correlates well with disease progression or recovery. The most immediate need regarding management is to establish the respective roles of surgery and antifungal therapy. Non-invasive disease requires surgical debridement and sinus ventilation only, though, additional oral or local corticosterold therapy may be beneficial in allergie type. For invasive disease, the adjuvant medical therapy is recommended to prevent recurrence and further extension. Itraconazole has been found as an effective drug in such situation. Patients with acute neerotizing type require radical surgery and amphotericin B therapy.

  2. [Emerging invasive fungal infections].

    PubMed

    Alvez, F; Figueras, C; Roselló, E

    2010-07-01

    The frequency and diversity of invasive fungal infections has changed over the last 25 years. The emergence of less common, but medically important fungi has increased, and the children at risk has expanded, with the inclusion of medical conditions such as cancer, mainly haematological malignancy or stem cell transplant, immunosuppressive therapy, prolonged neutropenia, and T-cell immunodeficiency. Among mould infections, fusariosis and phaeohyphomycosis (Dematiaceous fungi) have been increasingly reported in this group of patients. To successfully manage these challenging infections, it is imperative that paediatricians and sub-specialists remain aware of the optimal and timely diagnosis and therapeutic options. Unlike other common mycoses that cause human disease, there no simple antigen or serological tests available to detect these pathogens in tissue or blood. The outcome for these disseminate, and often refractory fungal infections in neutropenic patients and transplant recipients remains extremely poor, requiring early and aggressive therapy. Unfortunately there are no guidelines outlining the choices for optimal therapy in the treatment of paediatric invasive fungal infections do not exist, and on the other hand are limited paediatric data available comparing antifungal agents in children with proven, probable or suspected invasive fungal infection. The options for treatment rest mainly on some adult guidelines that comment on the treatment of these emerging and uncommon important fungi in children. Despite the sparse clinical trials available on treatment and its poor outcome, options for treatment of invasive fungal infections have increased with the advance of new antifungal agents, with improved tolerability and increased range of activity. The epidemiology, clinical manifestations, diagnosis and treatment of fusariosis and phaeohyphomycosis are discussed in this article.

  3. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

  4. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    PubMed Central

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-01-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface. PMID:26955973

  5. Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Fan, Yanhua; Pei, Xiaoqiong; Guo, Shujuan; Zhang, Yongjun; Luo, Zhibing; Liao, Xinggang; Pei, Yan

    2010-12-01

    Insect cuticles consist mainly of interlinked networks of proteins and the highly insoluble polysaccharide, chitin. Entomopathogenic fungi, such as Beauveria bassiana, invade insects by direct penetration of host cuticles via the action of diverse hydrolases including proteases and chitinases coupled to mechanical pressure. In order to better target cuticle protein-chitin structures and accelerate penetration speed, a hybrid protease (CDEP-BmChBD) was constructed by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP-1, a subtilisin-like protease from B. bassiana. Compared to the wild-type, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was enhanced by including proteases, CDEP-1 or CDEP:BmChBD produced in Pichia pastoris, as an additive, however, the augment effect of CDEP:BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrate that rational design virulence factor is a potential strategy for strain improvement by genetic engineering. PMID:20674735

  6. Identification and characterization of alkaline serine protease from goat skin surface metagenome.

    PubMed

    Pushpam, Paul Lavanya; Rajesh, Thangamani; Gunasekaran, Paramasamy

    2011-01-01

    Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively. PMID:21906326

  7. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    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. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  9. Cysteine protease of the nematode Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1 antibody response in rats.

    PubMed Central

    Kamata, I; Yamada, M; Uchikawa, R; Matsuda, S; Arizono, N

    1995-01-01

    Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pI 8.5, showed maximum enzyme activity at pH 5.5 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64, completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis, a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and IgG1, production of anti-cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response. Images Fig. 2 PMID:7554403

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

    PubMed Central

    2011-01-01

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

  11. Polyglycine hydrolases: Fungal β-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    PubMed Central

    Naumann, Todd A; Naldrett, Michael J; Ward, Todd J; Price, Neil P J

    2015-01-01

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochliobolus carbonum (Bz-cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es-cmp were analyzed by LC-MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β-lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β-lactamase-like region. This is the first report of a predicted β-lactamase that is an endoprotease. PMID:25966977

  12. Exogenous proteases for meat tenderization.

    PubMed

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  13. Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro.

    PubMed

    Chauhan, V; Sheikh, A M; Chauhan, A; Spivack, W D; Fenko, M D; Malik, M N

    2005-04-01

    The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40-75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP2) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP2 inhibited the enzyme (16%). The inhibition of the protease by PIP2 was concentration-dependent. During receptor-coupled cell activation, phospholipase A2 (PLA2) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP2 by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP2 to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA2, PI 4-kinase, PIP 5-kinase and PLC are involved. PMID:16010981

  14. Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

    SciTech Connect

    Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.; Ghassemian, Majid; Bandeira, Nuno; Komives, Elizabeth A.

    2014-03-01

    Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. By combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.

  15. Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots.

    PubMed

    Viterbo, Ada; Harel, Michal; Chet, Ilan

    2004-09-01

    Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.

  16. Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection[W][OPEN

    PubMed Central

    Blümke, Antje; Falter, Christian; Herrfurth, Cornelia; Sode, Björn; Bode, Rainer; Schäfer, Wilhelm; Feussner, Ivo; Voigt, Christian A.

    2014-01-01

    The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection. PMID:24686113

  17. Fungal diseases of horses.

    PubMed

    Cafarchia, Claudia; Figueredo, Luciana A; Otranto, Domenico

    2013-11-29

    Among diseases of horses caused by fungi (=mycoses), dermatophytosis, cryptococcosis and aspergillosis are of particular concern, due their worldwide diffusion and, for some of them, zoonotic potential. Conversely, other mycoses such as subcutaneous (i.e., pythiosis and mycetoma) or deep mycoses (i.e., blastomycosis and coccidioidomycosis) are rare, and/or limited to restricted geographical areas. Generally, subcutaneous and deep mycoses are chronic and progressive diseases; clinical signs include extensive, painful lesions (not pathognomonic), which resemble to other microbial infections. In all cases, early diagnosis is crucial in order to achieve a favorable prognosis. Knowledge of the epidemiology, clinical signs, and diagnosis of fungal diseases is essential for the establishment of effective therapeutic strategies. This article reviews the clinical manifestations, diagnosis and therapeutic protocols of equine fungal infections as a support to early diagnosis and application of targeted therapeutic and control strategies. PMID:23428378

  18. Fungal echinocandin resistance

    PubMed Central

    Walker, Louise A.; Gow, Neil A.R.; Munro, Carol A.

    2010-01-01

    The echinocandins are the newest class of antifungal agents in the clinical armory. These secondary metabolites are non-competitive inhibitors of the synthesis of β-(1,3)-glucan, a major structural component of the fungal cell wall. Recent work has shown that spontaneous mutations can arise in two hot spot regions of Fks1 the target protein of echinocandins that reduce the enzyme’s sensitivity to the drug. However, other strains have been isolated in which the sequence of FKS1 is unaltered yet the fungus has decreased sensitivity to echinocandins. In addition it has been shown that echinocandin-treatment can induce cell wall salvage mechanisms that result in the compensatory upregulation of chitin synthesis in the cell wall. This salvage mechanism strengthens cell walls damaged by exposure to echinocandins. Therefore, fungal resistance to echinocandins can arise due to the selection of either stable mutational or reversible physiological alterations that decrease susceptibility to these antifungal agents. PMID:19770064

  19. Fungal Sex and Pathogenesis

    PubMed Central

    Butler, Geraldine

    2010-01-01

    Summary: Human fungal pathogens are associated with diseases ranging from dandruff and skin colonization to invasive bloodstream infections. The major human pathogens belong to the Candida, Aspergillus, and Cryptococcus clades, and infections have high and increasing morbidity and mortality. Many human fungal pathogens were originally assumed to be asexual. However, recent advances in genome sequencing, which revealed that many species have retained the genes required for the sexual machinery, have dramatically influenced our understanding of the biology of these organisms. Predictions of a rare or cryptic sexual cycle have been supported experimentally for some species. Here, I examine the evidence that human pathogens reproduce sexually. The evolution of the mating-type locus in ascomycetes (including Candida and Aspergillus species) and basidiomycetes (Malassezia and Cryptococcus) is discussed. I provide an overview of how sex is suppressed in different species and discuss the potential associations with pathogenesis. PMID:20065328

  20. Fungal sex and pathogenesis.

    PubMed

    Butler, Geraldine

    2010-01-01

    Human fungal pathogens are associated with diseases ranging from dandruff and skin colonization to invasive bloodstream infections. The major human pathogens belong to the Candida, Aspergillus, and Cryptococcus clades, and infections have high and increasing morbidity and mortality. Many human fungal pathogens were originally assumed to be asexual. However, recent advances in genome sequencing, which revealed that many species have retained the genes required for the sexual machinery, have dramatically influenced our understanding of the biology of these organisms. Predictions of a rare or cryptic sexual cycle have been supported experimentally for some species. Here, I examine the evidence that human pathogens reproduce sexually. The evolution of the mating-type locus in ascomycetes (including Candida and Aspergillus species) and basidiomycetes (Malassezia and Cryptococcus) is discussed. I provide an overview of how sex is suppressed in different species and discuss the potential associations with pathogenesis. PMID:20065328

  1. Small intestinal fungal overgrowth.

    PubMed

    Erdogan, Askin; Rao, Satish S C

    2015-04-01

    Small intestinal fungal overgrowth (SIFO) is characterized by the presence of excessive number of fungal organisms in the small intestine associated with gastrointestinal (GI) symptoms. Candidiasis is known to cause GI symptoms particularly in immunocompromised patients or those receiving steroids or antibiotics. However, only recently, there is emerging literature that an overgrowth of fungus in the small intestine of non-immunocompromised subjects may cause unexplained GI symptoms. Two recent studies showed that 26 % (24/94) and 25.3 % (38/150) of a series of patients with unexplained GI symptoms had SIFO. The most common symptoms observed in these patients were belching, bloating, indigestion, nausea, diarrhea, and gas. The underlying mechanism(s) that predisposes to SIFO is unclear but small intestinal dysmotility and use of proton pump inhibitors has been implicated. However, further studies are needed; both to confirm these observations and to examine the clinical relevance of fungal overgrowth, both in healthy subjects and in patients with otherwise unexplained GI symptoms. Importantly, whether eradication or its treatment leads to resolution of symptoms remains unclear; at present, a 2-3-week course of antifungal therapy is recommended and may be effective in improving symptoms, but evidence for eradication is lacking. PMID:25786900

  2. Developments in Fungal Taxonomy

    PubMed Central

    Guarro, Josep; Gené, Josepa; Stchigel, Alberto M.

    1999-01-01

    Fungal infections, especially those caused by opportunistic species, have become substantially more common in recent decades. Numerous species cause human infections, and several new human pathogens are discovered yearly. This situation has created an increasing interest in fungal taxonomy and has led to the development of new methods and approaches to fungal biosystematics which have promoted important practical advances in identification procedures. However, the significance of some data provided by the new approaches is still unclear, and results drawn from such studies may even increase nomenclatural confusion. Analyses of rRNA and rDNA sequences constitute an important complement of the morphological criteria needed to allow clinical fungi to be more easily identified and placed on a single phylogenetic tree. Most of the pathogenic fungi so far described belong to the kingdom Fungi; two belong to the kingdom Chromista. Within the Fungi, they are distributed in three phyla and in 15 orders (Pneumocystidales, Saccharomycetales, Dothideales, Sordariales, Onygenales, Eurotiales, Hypocreales, Ophiostomatales, Microascales, Tremellales, Poriales, Stereales, Agaricales, Schizophyllales, and Ustilaginales). PMID:10398676

  3. Fungal toenail infections

    PubMed Central

    2014-01-01

    Introduction Fungal infections are reported to cause 23% of foot diseases and 50% of nail conditions in people seen by dermatologists, but are less common in the general population, affecting 3% to 12% of people. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of oral treatments for fungal toenail infections in adults? What are the effects of topical treatments for fungal toenail infections in adults? We searched: Medline, Embase, The Cochrane Library, and other important databases up to October 2013 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 13 studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review, we present information relating to the effectiveness and safety of the following interventions: amorolfine, butenafine, ciclopirox, fluconazole, itraconazole, terbinafine, tioconazole, and topical ketoconazole. PMID:24625577

  4. Fungal toenail infections

    PubMed Central

    2011-01-01

    Introduction Fungal infections are reported to cause 23% of foot diseases and 50% of nail conditions in people seen by dermatologists, but are less common in the general population, affecting 3% to 5% of people. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of oral treatments for fungal toenail infections? What are the effects of topical treatments for fungal toenail infections? We searched: Medline, Embase, The Cochrane Library, and other important databases up to March 2011 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 12 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review we present information relating to the effectiveness and safety of the following interventions: amorolfine, butenafine, ciclopirox, fluconazole, griseofulvin, itraconazole, ketoconazole, mechanical debridement, terbinafine, and tioconazole. PMID:21846413

  5. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  6. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    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. PMID:24832807

  7. Cuticle-degrading proteases and toxins as virulence markers of Beauveria bassiana (Balsamo) Vuillemin.

    PubMed

    Cito, Annarita; Barzanti, Gian Paolo; Strangi, Agostino; Francardi, Valeria; Zanfini, Assunta; Dreassi, Elena

    2016-09-01

    Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle-degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro-in medium filtrates and mycelia-and in vivo-in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain-dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae. PMID:27198125

  8. Isolation of Pyrrolocins A–C: cis- and trans-Decalin Tetramic Acid Antibiotics from an Endophytic Fungal-Derived Pathway

    PubMed Central

    2014-01-01

    Three new decalin-type tetramic acid analogues, pyrrolocins A (1), B (2), and C (3), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced 1 but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, 1–3 were produced. All three compounds are structurally related to equisetin-type compounds, with 1 and 3 having a trans-decalin ring system, while 2 has a cis-fused decalin. All were active against Mycobacterium tuberculosis, with the trans-decalin analogues 1 and 3 exhibiting lower MICs than the cis-decalin analogue 2. Here we report the isolation, structure elucidation, and antimycobacterial activities of 1–3 from the recombinant expression as well as the isolation of 1 from the wild-type fungus NRRL 50135. PMID:25351193

  9. Intensification of biocatalytical processes by synergistic substrate conversion. Fungal peroxidase catalyzed N-hydroxy derivative oxidation in presence of 10-propyl sulfonic acid phenoxazine.

    PubMed

    Kulys, Juozas; Dapkunas, Zilvinas; Stupak, Robert

    2009-08-01

    Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 +/- 0.2).10(7) M(-1) s(-1) and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA-PPSA and HPCM-PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme-substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.

  10. Diagnostic of Fungal Infections Related to Biofilms.

    PubMed

    Sanguinetti, Maurizio; Posteraro, Brunella

    2016-01-01

    Fungal biofilm-related infections, most notably those caused by the Candida and Aspergillus genera, need to be diagnosed accurately and rapidly to avoid often unfavorable outcomes. Despite diagnosis of these infections is still based on the traditional histopathology and culture, the use of newer, rapid methods has enormously enhanced the diagnostic capability of a modern clinical mycology laboratory. Thus, while accurate species-level identification of fungal isolates can be achieved with turnaround times considerably shortened, nucleic acid-based or antigen-based detection methods can be considered useful adjuncts for the diagnosis of invasive forms of candidiasis and aspergillosis. Furthermore, simple, reproducible, and fast methods have been developed to quantify biofilm production by fungal isolates in vitro. In this end, isolates can be categorized as low, moderate, or high biofilm-forming, and this categorization may reflect their differential response to the conventional antifungal therapy. By means of drug susceptibility testing performed on fungal biofilm-growing isolates, it is now possible to evaluate not only the activity of conventional antifungal agents, but also of novel anti-biofilm agents. Despite this, future diagnostic methods need to target specific biofilm components/molecules, in order to provide a direct proof of the presence of this growth phenotype on the site of infection. In the meantime, our knowledge of the processes underlying the adaptive drug resistance within the biofilm has put into evidence biofilm-specific molecules that could be potentially helpful as therapeutic targets. Surely, the successful management of clinically relevant fungal biofilms will rely upon the advancement and/or refinement of these approaches. PMID:27300347

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

    PubMed

    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.

  12. Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

    PubMed Central

    Adam, Céline; Picard, Marguerite; Déquard-Chablat, Michelle; Sellem, Carole H.; Denmat, Sylvie Hermann-Le; Contamine, Véronique

    2012-01-01

    Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction. PMID:22693589

  13. Processing and targeting of the thiol protease aleurain: Progress report

    SciTech Connect

    Rogers, J.C.

    1988-01-01

    This study addresses the processing and targeting of the thiol protease aleurain in monocots. A probe derived from the aleurain cDNA specific for the 5'-most 400 bp (a region encoding the first 140 amino acids of the preprotein hybridized to at least 3 separate elements in the barley genome; only one represented the aleurain gene. In contrast, a probe specific for the remaining 2/23 of the cDNA (representing the protease domain) hybridized to only a single copy sequence. To know if this pattern pertained in other, closely related, monocots, we probed Southern blots of genomic DNA from maize, rye, oats, sorghum, and pearl millet with each probe. In each instance except for maize DNA, the 5' domain probe hybridizes to several fragments in addition to those identified by the protease domain probe. Presumable the darkest hybridization in each represents the fragment carrying the sequences homologous to barley aleurain. The fragments from a given restriction enzyme identified by the protease domain probe in sorghum, millet, and maize, were indistinguishable in size indicating that the gene sequences, as well as flanking DNA, are so well conserved among the group that the location of the hexanucleotide sequences have not diverged. (3 refs., 3 figs.)

  14. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

    PubMed

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

  15. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    PubMed Central

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W.

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

  16. Fungal osteomyelitis and septic arthritis.

    PubMed

    Bariteau, Jason T; Waryasz, Gregory R; McDonnell, Matthew; Fischer, Staci A; Hayda, Roman A; Born, Christopher T

    2014-06-01

    Management of fungal osteomyelitis and fungal septic arthritis is challenging, especially in the setting of immunodeficiency and conditions that require immunosuppression. Because fungal osteomyelitis and fungal septic arthritis are rare conditions, study of their pathophysiology and treatment has been limited. In the literature, evidence-based treatment is lacking and, historically, outcomes have been poor. The most common offending organisms are Candida and Aspergillus, which are widely distributed in humans and soil. However, some fungal pathogens, such as Histoplasma, Blastomyces, Coccidioides, Cryptococcus, and Sporothrix, have more focal areas of endemicity. Fungal bone and joint infections result from direct inoculation, contiguous infection spread, or hematogenous seeding of organisms. These infections may be difficult to diagnose and eradicate, especially in the setting of total joint arthroplasty. Although there is no clear consensus on treatment, guidelines are available for management of many of these pathogens.

  17. 454-Pyrosequencing Reveals Variable Fungal Diversity Across Farming Systems

    PubMed Central

    Kazeeroni, Elham A.; Al-Sadi, Abdullah M.

    2016-01-01

    Oasis farming system is common in some parts of the world, especially in the Arabian Peninsula and several African countries. In Oman, the farming system in the majority of farms follows a semi-oasis farming (SOF) system, which is characterized by growing multiple crops mainly for home consumption, but also for local market. This study was conducted to investigate fungal diversity using pyrosequencing approach in soils from a farm utilizing a SOF system which is cultivated with date palms, acid limes and cucumbers. Fungal diversity from this farm was compared to that from an organic farm (OR) growing cucumbers and tomatoes. Fungal diversity was found to be variable among different crops in the same farm. The observed OTUs, Chao1 richness estimates and Shannon diversity values indicated that soils from date palms and acid limes have higher fungal diversity compared to soil from cucumbers (SOF). In addition, they also indicated that the level of fungal diversity is higher in the rhizosphere of cucumbers grown in OR compared to SOF. Ascomycota was the most dominant phylum in most of the samples from the OR and SOF farms. Other dominant phyla are Microsporidia, Chytridiomycota, and Basidiomycota. The differential level of fungal diversity within the SOF could be related to the variation in the cultural practices employed for each crop. PMID:27014331

  18. 454-Pyrosequencing Reveals Variable Fungal Diversity Across Farming Systems.

    PubMed

    Kazeeroni, Elham A; Al-Sadi, Abdullah M

    2016-01-01

    Oasis farming system is common in some parts of the world, especially in the Arabian Peninsula and several African countries. In Oman, the farming system in the majority of farms follows a semi-oasis farming (SOF) system, which is characterized by growing multiple crops mainly for home consumption, but also for local market. This study was conducted to investigate fungal diversity using pyrosequencing approach in soils from a farm utilizing a SOF system which is cultivated with date palms, acid limes and cucumbers. Fungal diversity from this farm was compared to that from an organic farm (OR) growing cucumbers and tomatoes. Fungal diversity was found to be variable among different crops in the same farm. The observed OTUs, Chao1 richness estimates and Shannon diversity values indicated that soils from date palms and acid limes have higher fungal diversity compared to soil from cucumbers (SOF). In addition, they also indicated that the level of fungal diversity is higher in the rhizosphere of cucumbers grown in OR compared to SOF. Ascomycota was the most dominant phylum in most of the samples from the OR and SOF farms. Other dominant phyla are Microsporidia, Chytridiomycota, and Basidiomycota. The differential level of fungal diversity within the SOF could be related to the variation in the cultural practices employed for each crop. PMID:27014331

  19. 454-Pyrosequencing Reveals Variable Fungal Diversity Across Farming Systems.

    PubMed

    Kazeeroni, Elham A; Al-Sadi, Abdullah M

    2016-01-01

    Oasis farming system is common in some parts of the world, especially in the Arabian Peninsula and several African countries. In Oman, the farming system in the majority of farms follows a semi-oasis farming (SOF) system, which is characterized by growing multiple crops mainly for home consumption, but also for local market. This study was conducted to investigate fungal diversity using pyrosequencing approach in soils from a farm utilizing a SOF system which is cultivated with date palms, acid limes and cucumbers. Fungal diversity from this farm was compared to that from an organic farm (OR) growing cucumbers and tomatoes. Fungal diversity was found to be variable among different crops in the same farm. The observed OTUs, Chao1 richness estimates and Shannon diversity values indicated that soils from date palms and acid limes have higher fungal diversity compared to soil from cucumbers (SOF). In addition, they also indicated that the level of fungal diversity is higher in the rhizosphere of cucumbers grown in OR compared to SOF. Ascomycota was the most dominant phylum in most of the samples from the OR and SOF farms. Other dominant phyla are Microsporidia, Chytridiomycota, and Basidiomycota. The differential level of fungal diversity within the SOF could be related to the variation in the cultural practices employed for each crop.

  20. Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato.

    PubMed

    Abbasi, P A; Lazarovits, G

    2006-10-01

    Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3-2.6, oxidation-reduction potential 1007-1025 mV, and free active chlorine concentration 27-35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4-8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide. PMID:17110959

  1. Type II Transmembrane Serine Proteases*

    PubMed Central

    Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

    2009-01-01

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  2. Fungal mastoiditis in immunocompromised children.

    PubMed

    Slack, C L; Watson, D W; Abzug, M J; Shaw, C; Chan, K H

    1999-01-01

    The immunocompromised host is subject to a variety of opportunistic infections. Mycotic infections, including invasive fungal sinusitis, are a dreaded complication in immune deficient children. Fungal mastoiditis has rarely been described in this population. Our experience with 2 cases of fungal mastoiditis in immunocompromised children is reviewed. Case histories describing aggressive medical management with and without surgical intervention and a review of the literature are presented. PMID:9932592

  3. Fungal Genome Sequencing and Bioenergy

    SciTech Connect

    Baker, Scott E.; Thykaer, Jette; Adney, William S.; Brettin, T.; Brockman, Fred J.; D'haeseleer, Patrik; Martinez, Antonio D.; Miller, R. M.; Rokhsar, Daniel S.; Schadt, Christopher W.; Torok, Tamas; Tuskan, Gerald; Bennett, Joan W.; Berka, Randy; Briggs, Steve; Heitman, Joseph; Taylor, John; Turgeon, Barbara G.; Werner-Washburne, Maggie; Himmel, Michael E.

    2008-09-30

    To date, the number of ongoing filamentous fungal genome sequencing projects is almost tenfold fewer than those of bacterial and archaeal genome projects. The fungi chosen for sequencing represent narrow kingdom diversity; most are pathogens or models. We advocate an ambitious, forward-looking phylogenetic-based genome sequencing program, designed to capture metabolic diversity within the fungal kingdom, thereby enhancing research into alternative bioenergy sources, bioremediation, and fungal-environment interactions.

  4. Fungal Genome Sequencing and Bioenergy

    SciTech Connect

    Baker, Scott; Thykaer, Jette; Adney, William S; Brettin, Tom; Brockman, Fred; Dhaeseleer, Patrick; Martinez, A diego; Miller, R michael; Rokhsar, Daniel; Schadt, Christopher Warren; Torok, Tamas; Tuskan, Gerald A; Bennett, Joan; Berka, Randy; Briggs, Steven; Heitman, Joseph; Taylor, John; Turgeon, Gillian; Werner-Washburne, Maggie; Himmel, Michael E

    2008-01-01

    To date, the number of ongoing filamentous fungal genome sequencing projects is almost tenfold fewer than those of bacterial and archaeal genome projects. The fungi chosen for sequencing represent narrow kingdom diversity; most are pathogens or models. We advocate an ambitious, forward-looking phylogenetic-based genome sequencing program, designed to capture metabolic diversity within the fungal kingdom, thereby enhancing research into alternative bioenergy sources, bioremediation, and fungal-environment interactions. Published by Elsevier Ltd on behalf of The British Mycological Society.

  5. Fungal Genome Sequencing and Bioenergy

    SciTech Connect

    Schadt, Christopher Warren; Baker, Scott; Thykaer, Jette; Adney, William S; Brettin, Tom; Brockman, Fred; Dhaeseleer, Patrick; Martinez, A diego; Miller, R michael; Rokhsar, Daniel; Torok, Tamas; Tuskan, Gerald A; Bennett, Joan; Berka, Randy; Briggs, Steven; Heitman, Joseph; Rizvi, L; Taylor, John; Turgeon, Gillian; Werner-Washburne, Maggie; Himmel, Michael

    2008-01-01

    To date, the number of ongoing filamentous fungal genome sequencing projects is almost tenfold fewer than those of bacterial and archaeal genome projects. The fungi chosen for sequencing represent narrow kingdom diversity; most are pathogens or models. We advocate an ambitious, forward-looking phylogenetic-based genome sequencing program, designed to capture metabolic diversity within the fungal kingdom, thereby enhancing research into alternative bioenergy sources, bioremediation, and fungal-environment interactions.

  6. Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens.

    PubMed

    Park, Hyun Bong; Crawford, Jason M

    2016-08-01

    Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm. PMID:27353165

  7. Molecular Imaging of Proteases in Cancer

    PubMed Central

    Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction. PMID:20234801

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

    SciTech Connect

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

    1987-06-16

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

  9. Evolutionary Analysis of Novel Serine Proteases in the Venom Gland Transcriptome of Bitis gabonica rhinoceros

    PubMed Central

    Vaiyapuri, Sakthivel; Wagstaff, Simon C.; Harrison, Robert A.; Gibbins, Jonathan M.; Hutchinson, E. Gail

    2011-01-01

    Background Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites. PMID:21731776

  10. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast.

    PubMed Central

    Schneider, A; Behrens, M; Scherer, P; Pratje, E; Michaelis, G; Schatz, G

    1991-01-01

    Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer. This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum. Images PMID:1991446

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

    PubMed

    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. PMID:23082957

  12. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity

    PubMed Central

    Herod, Morgan R.; Prince, Cynthia A.; Skilton, Rachel J.; Ward, Vernon K.; Cooper, Jonathan B.; Clarke, Ian N.

    2014-01-01

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

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

    PubMed

    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.

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

    PubMed Central

    Lomate, Purushottam R.; Bonning, Bryony C.

    2016-01-01

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

  15. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.

    PubMed

    Herod, Morgan R; Prince, Cynthia A; Skilton, Rachel J; Ward, Vernon K; Cooper, Jonathan B; Clarke, Ian N

    2014-12-15

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  16. Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

    PubMed

    Shivanand, Pooja; Jayaraman, Gurunathan

    2011-04-01

    A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

  17. Involvement of Abscisic Acid in the Coordinated Regulation of a Stress-Inducible Hexose Transporter (VvHT5) and a Cell Wall Invertase in Grapevine in Response to Biotrophic Fungal Infection[W

    PubMed Central

    Hayes, Matthew A.; Feechan, Angela; Dry, Ian B.

    2010-01-01

    Biotrophic fungal and oomycete pathogens alter carbohydrate metabolism in infected host tissues. Symptoms such as elevated soluble carbohydrate concentrations and increased invertase activity suggest that a pathogen-induced carbohydrate sink is established. To identify pathogen-induced regulators of carbohydrate sink strength, quantitative real-time polymerase chain reaction was used to measure transcript levels of invertase and hexose transporter genes in biotrophic pathogen-infected grapevine (Vitis vinifera) leaves. The hexose transporter VvHT5 was highly induced in coordination with the cell wall invertase gene VvcwINV by powdery and downy mildew infection. However, similar responses were also observed in response to wounding, suggesting that this is a generalized response to stress. Analysis of the VvHT5 promoter region indicated the presence of multiple abscisic acid (ABA) response elements, suggesting a role for ABA in the transition from source to sink under stress conditions. ABA treatment of grape leaves was found to reproduce the same gene-specific transcriptional changes as observed under biotic and abiotic stress conditions. Furthermore, the key regulatory ABA biosynthetic gene, VvNCED1, was activated under these same stress conditions. VvHT5 promoter::β-glucuronidase-directed expression in transgenic Arabidopsis (Arabidopsis thaliana) was activated by infection with powdery mildew and by ABA treatment, and the expression was closely associated with vascular tissue adjacent to infected regions. Unlike VvHT1 and VvHT3, which appear to be predominantly involved in hexose transport in developing leaves and berries, VvHT5 appears to have a specific role in enhancing sink strength under stress conditions, and this is controlled through ABA. Our data suggest a central role for ABA in the regulation of VvcwINV and VvHT5 expression during the transition from source to sink in response to infection by biotrophic pathogens. PMID:20348211

  18. Molecular cloning and sequence determination of the genomic regions encoding protease and genome-linked protein of three picornaviruses.

    PubMed Central

    Werner, G; Rosenwirth, B; Bauer, E; Seifert, J M; Werner, F J; Besemer, J

    1986-01-01

    To investigate the degree of similarity between picornavirus proteases, we cloned the genomic cDNAs of an enterovirus, echovirus 9 (strain Barty), and two rhinoviruses, serotypes 1A and 14LP, and determined the nucleotide sequence of the region which, by analogy to poliovirus, encodes the protease. The nucleotide sequence of the region encoding the genome-linked protein VPg, immediately adjacent to the protease, was also determined. Comparison of nucleotide and deduced amino acid sequences with other available picornavirus sequences showed remarkable homology in proteases and among VPgs. Three highly conserved peptide regions were identified in the protease; one of these is specific for human picornaviruses and has no obvious counterpart in encephalomyocarditis virus, foot-and-mouth disease virus, or cowpea mosaic virus proteases. Within the other two peptide regions two conserved amino acids, Cys 147 and His 161, could be the reactive residues of the active site. We used a statistical method to predict certain features of the secondary structures, such as alpha helices, beta sheets, and turns, and found many of these conformations to be conserved. The hydropathy profiles of the compared proteases were also strikingly similar. Thus, the proteases of human picornaviruses very probably have a similar three-dimensional structure. Images PMID:3512851

  19. Fungal biodiversity to biotechnology.

    PubMed

    Chambergo, Felipe S; Valencia, Estela Y

    2016-03-01

    Fungal habitats include soil, water, and extreme environments. With around 100,000 fungus species already described, it is estimated that 5.1 million fungus species exist on our planet, making fungi one of the largest and most diverse kingdoms of eukaryotes. Fungi show remarkable metabolic features due to a sophisticated genomic network and are important for the production of biotechnological compounds that greatly impact our society in many ways. In this review, we present the current state of knowledge on fungal biodiversity, with special emphasis on filamentous fungi and the most recent discoveries in the field of identification and production of biotechnological compounds. More than 250 fungus species have been studied to produce these biotechnological compounds. This review focuses on three of the branches generally accepted in biotechnological applications, which have been identified by a color code: red, green, and white for pharmaceutical, agricultural, and industrial biotechnology, respectively. We also discuss future prospects for the use of filamentous fungi in biotechnology application.

  20. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  1. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  2. Biased Signaling of Protease-Activated Receptors

    PubMed Central

    Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions. PMID:24860547

  3. Managing acute invasive fungal sinusitis.

    PubMed

    Dwyhalo, Kristina M; Donald, Carrlene; Mendez, Anthony; Hoxworth, Joseph

    2016-01-01

    Acute invasive fungal sinusitis is the most aggressive form of fungal sinusitis and can be fatal, especially in patients who are immunosuppressed. Early diagnosis and intervention are crucial and potentially lifesaving, so primary care providers must maintain a high index of suspicion for this disease. Patients may need to be admitted to the hospital for IV antifungal therapy and surgical debridement.

  4. An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus.

    PubMed

    Chen, F R; Liu, P C; Lee, K K

    1999-01-01

    Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease. PMID:10413876

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

    PubMed

    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.

  6. Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis.

    PubMed Central

    Koide, Y; Nakamura, A; Uozumi, T; Beppu, T

    1986-01-01

    A Bacillus subtilis 2.7-kilobase DNA fragment containing an intracellular protease gene was cloned into Escherichia coli. The transformants produced an intracellular protease of approximately 35,000 Mr whose activity was inhibited by both phenylmethylsulfonyl fluoride and EDTA. Introduction of the fragment on a multicopy vector, pUB110, into B. subtilis caused a marked increase in the level of the intracellular protease. The nucleotide sequence of the cloned fragment showed the presence of an open reading frame for a possible proenzyme of the major intracellular serine protease (ISP-I) of B. subtilis with an NH2-terminal 17- or 20-amino-acid extension. The total amino acid sequence of the protease deduced from the nucleotide sequence showed considerable homology with that of an extracellular serine protease, subtilisin. The transcriptional initiation site of the ISP-I gene was identified by nuclease S1 mapping. No typical conserved sequence for promoters was found upstream of the open reading frame. An ISP-I-negative mutant of B. subtilis was constructed by integration of artificially deleted gene into the chromosome. The mutant sporulated normally in a nutritionally rich medium but showed decreased sporulation in a synthetic medium. The chloramphenicol resistance determinant of a plasmid integrated at the ISP-I locus was mapped by PBS1 transduction and was found to be closely linked to metC (99.5%). Images PMID:3087947

  7. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

    PubMed Central

    Bagdasarian, A; Wheeler, J; Stewart, G J; Ahmed, S S; Colman, R W

    1981-01-01

    Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin. Images PMID:6161137

  10. Transcriptional and Proteomic Analysis of the Aspergillus fumigatus ΔprtT Protease-Deficient Mutant

    PubMed Central

    Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W. P.; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus. PMID:22514608

  11. Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit1[C][W][OA

    PubMed Central

    Nieuwenhuizen, Niels J.; Maddumage, Ratnasiri; Tsang, Gianna K.; Fraser, Lena G.; Cooney, Janine M.; De Silva, H. Nihal; Green, Sol; Richardson, Kim A.; Atkinson, Ross G.

    2012-01-01

    Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin. PMID:22039217

  12. Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

    PubMed

    Smid, Ida; Gruden, Kristina; Buh Gašparič, Meti; Koruza, Katarina; Petek, Marko; Pohleven, Jure; Brzin, Jože; Kos, Janko; Zel, Jana; Sabotič, Jerica

    2013-12-26

    Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests. PMID:24295324

  13. Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

    PubMed

    Smid, Ida; Gruden, Kristina; Buh Gašparič, Meti; Koruza, Katarina; Petek, Marko; Pohleven, Jure; Brzin, Jože; Kos, Janko; Zel, Jana; Sabotič, Jerica

    2013-12-26

    Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

  14. Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms.

    PubMed

    Carrillo, Laura; Herrero, Ignacio; Cambra, Inés; Sánchez-Monge, Rosa; Diaz, Isabel; Martinez, Manuel

    2011-10-01

    Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed. PMID:21482127

  15. Enzymatic bioremediation of polyaromatic hydrocarbons by fungal consortia enriched from petroleum contaminated soil and oil seeds.

    PubMed

    Balaji, V; Arulazhagan, P; Ebenezer, P

    2014-05-01

    The present study focuses on fungal strains capable of secreting extracellular enzymes by utilizing hydrocarbons present in the contaminated soil. Fungal strains were enriched from petroleum hydrocarbons contaminated soil samples collected from Chennai city, India. The potential fungi were isolated and screened for their enzyme secretion such as lipase, laccase, peroxidase and protease and also evaluated fungal enzyme mediated PAHs degradation. Total, 21 potential PAHs degrading fungi were isolated from PAHs contaminated soil, which belongs to 9 genera such as Aspergillus, Curvularia, Drechslera, Fusarium, Lasiodiplodia, Mucor Penicillium, Rhizopus, Trichoderma, and two oilseed-associated fungal genera such as Colletotrichum and Lasiodiplodia were used to test their efficacy in degradation of PAHs in polluted soil. Maximum lipase production was obtained with P. chrysogenum, M. racemosus and L. theobromae VBE1 under optimized cultural condition, which utilized PAHs in contaminated soil as sole carbon source. Fungal strains, P. chrysogenum, M. racemosus and L. theobromae VBE1, as consortia, used in the present study were capable of degrading branched alkane isoprenoids such as pristine (C17) and pyrene (C18) present in PAHs contaminated soil with high lipase production. The fungal consortia acts as potential candidate for bioremediation of PAHs contaminated environments.

  16. Enzymatic bioremediation of polyaromatic hydrocarbons by fungal consortia enriched from petroleum contaminated soil and oil seeds.

    PubMed

    Balaji, V; Arulazhagan, P; Ebenezer, P

    2014-05-01

    The present study focuses on fungal strains capable of secreting extracellular enzymes by utilizing hydrocarbons present in the contaminated soil. Fungal strains were enriched from petroleum hydrocarbons contaminated soil samples collected from Chennai city, India. The potential fungi were isolated and screened for their enzyme secretion such as lipase, laccase, peroxidase and protease and also evaluated fungal enzyme mediated PAHs degradation. Total, 21 potential PAHs degrading fungi were isolated from PAHs contaminated soil, which belongs to 9 genera such as Aspergillus, Curvularia, Drechslera, Fusarium, Lasiodiplodia, Mucor Penicillium, Rhizopus, Trichoderma, and two oilseed-associated fungal genera such as Colletotrichum and Lasiodiplodia were used to test their efficacy in degradation of PAHs in polluted soil. Maximum lipase production was obtained with P. chrysogenum, M. racemosus and L. theobromae VBE1 under optimized cultural condition, which utilized PAHs in contaminated soil as sole carbon source. Fungal strains, P. chrysogenum, M. racemosus and L. theobromae VBE1, as consortia, used in the present study were capable of degrading branched alkane isoprenoids such as pristine (C17) and pyrene (C18) present in PAHs contaminated soil with high lipase production. The fungal consortia acts as potential candidate for bioremediation of PAHs contaminated environments. PMID:24813008

  17. Maize death acids, 9-lipoxygenase-derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators.

    PubMed

    Christensen, Shawn A; Huffaker, Alisa; Kaplan, Fatma; Sims, James; Ziemann, Sebastian; Doehlemann, Gunther; Ji, Lexiang; Schmitz, Robert J; Kolomiets, Michael V; Alborn, Hans T; Mori, Naoki; Jander, Georg; Ni, Xinzhi; Sartor, Ryan C; Byers, Sara; Abdo, Zaid; Schmelz, Eric A

    2015-09-01

    Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed "jasmonates." As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed "death acids." 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions. PMID:26305953

  18. Maize death acids, 9-lipoxygenase–derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators

    PubMed Central

    Christensen, Shawn A.; Huffaker, Alisa; Kaplan, Fatma; Sims, James; Ziemann, Sebastian; Doehlemann, Gunther; Ji, Lexiang; Schmitz, Robert J.; Kolomiets, Michael V.; Alborn, Hans T.; Mori, Naoki; Jander, Georg; Ni, Xinzhi; Sartor, Ryan C.; Byers, Sara; Abdo, Zaid; Schmelz, Eric A.

    2015-01-01

    Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed “jasmonates.” As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed “death acids.” 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions. PMID:26305953

  19. Occurrence of aspartyl proteases in brine after herring marinating.

    PubMed

    Szymczak, Mariusz; Lepczyński, Adam

    2016-03-01

    Herrings are marinated in a brine consisting of salt and acetic acid. During marinating, various nitrogen fractions diffuse from fish flesh to the brine, causing significant nutritional quality losses of the raw material. In this study, it has been demonstrated for the first time that proteases diffuse from the fish to the marinating brine. Using ammonium sulphate precipitation and affinity chromatography on pepstatin-A agarose bed the aspartyl proteases were purified and concentrated over 2600-fold from a marinating brine. Pepstatin-A completely inhibited the activity of the purified preparation. The preparation was active against fluorogenic substrates specific for cathepsin D and E and inactive against substrates specific for cysteine cathepsins. Depending on incubation time, the preparation showed pH-optimum at 2.0 or 4.5. The 2D SDS-PAGE separation demonstrated the presence of a few proteins with molecular weights and pI values typical of cathepsin D, E and pepsin.

  20. Fungal osteomyelitis of maxillofacial bones: Rare presentation

    PubMed Central

    Urs, Aadithya B; Singh, Hanspal; Mohanty, Sujata; Sharma, Pankaj

    2016-01-01

    Osteomyelitis is defined as inflammation of the medullary cavities, haversian system and adjacent cortex of bone. Osteomyelitis involving maxilla is quite rare when compared to that of mandible. Fungal osteomyelitis is very rarely seen and documented in the maxillofacial area. It is devastating to patients if it is invasive in nature. A prospective study was undertaken from December 2011 to December 2013. Biopsied hard tissue bits were decalcified and sections were stained with H&E, periodic acid Schiff and Grocott methenamine silver. Culture sensitivity was carried out in all cases. Surgical intervention followed by antifungal therapy was advocated to treat the patients. The current series showed five cases of fungal osteomyelitis which included candidal osteomyelitis, aspergillosis and mucormycosis with slight predominance of maxilla over mandible. Osteomyelitis of facial bones needs to be investigated thoroughly as there is no difference in clinical presentation between bacterial and fungal osteomyelitis. The diagnostic workup with biopsy and culture sensitivity helps to identify the pathogen at the earliest. Appropriate antifungal treatment needs to be advocated for successful treatment. PMID:27721629

  1. New strategic insights into managing fungal biofilms

    PubMed Central

    Borghi, Elisa; Morace, Giulia; Borgo, Francesca; Rajendran, Ranjith; Sherry, Leighann; Nile, Christopher; Ramage, Gordon

    2015-01-01

    Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation, or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e., chronic inflammation and biofilm formation, could represent an advantage in the clinical setting. In this paper we discuss the antifungal properties of myriocin, fulvic acid, and acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections. PMID:26500623

  2. Fungal peroxisomes as biosynthetic organelles.

    PubMed

    Stehlik, Thorsten; Sandrock, Björn; Ast, Julia; Freitag, Johannes

    2014-12-01

    Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the β-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example β-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes.

  3. Development of a glutathione production process from proteinaceous biomass resources using protease-displaying Saccharomyces cerevisiae.

    PubMed

    Hara, Kiyotaka Y; Kim, Songhee; Yoshida, Hideyo; Kiriyama, Kentaro; Kondo, Takashi; Okai, Naoko; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2012-02-01

    Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources. PMID:22075633

  4. Antifungal effects of undecylenic acid on the biofilm formation of Candida albicans.

    PubMed

    Shi, Dongmei; Zhao, Yaxin; Yan, Hongxia; Fu, Hongjun; Shen, Yongnian; Lu, Guixia; Mei, Huan; Qiu, Ying; Li, Dongmei; Liu, Weida

    2016-05-01

    Undecylenic acid can effectively control skin fungal infection, but the mechanism of its fungal inhibition is unclear. Hyphal growth of Candida albicans (C. albicans) and biofilm formation have been well recognized as important virulence factors for the initiation of skin infection and late development of disseminated infection. In this study, we seek to investigate antifungal mechanisms of undecylenic acid by evaluating the virulence factors of C. albicans during biofilm formation. We found that undecylenic acid inhibits biofilm formation of C. albicans effectively with optimal concentration above 3 mM. In the presence of this compound, the morphological transition from yeast to filamentous phase is abolished ultimately when the concentration of undecylenic acid is above 4 mM. Meanwhile, the cell surface is crumpled, and cells display an atrophic appearance under scanning electron microscopy even with low concentration of drug treatment. On the other hand, the drug treatment decreases the transcriptions of hydrolytic enzymes such as secreted aspartic protease, lipase, and phospholipase. Hyphal formation related genes, like HWP1, are significantly reduced in transcriptional level in drug-treated biofilm condition as well. The down-regulated profile of these genes leads to a poorly organized biofilm in undecylenic acid treated environment.

  5. Antifungal effects of undecylenic acid on the biofilm formation of Candida albicans.

    PubMed

    Shi, Dongmei; Zhao, Yaxin; Yan, Hongxia; Fu, Hongjun; Shen, Yongnian; Lu, Guixia; Mei, Huan; Qiu, Ying; Li, Dongmei; Liu, Weida

    2016-05-01

    Undecylenic acid can effectively control skin fungal infection, but the mechanism of its fungal inhibition is unclear. Hyphal growth of Candida albicans (C. albicans) and biofilm formation have been well recognized as important virulence factors for the initiation of skin infection and late development of disseminated infection. In this study, we seek to investigate antifungal mechanisms of undecylenic acid by evaluating the virulence factors of C. albicans during biofilm formation. We found that undecylenic acid inhibits biofilm formation of C. albicans effectively with optimal concentration above 3 mM. In the presence of this compound, the morphological transition from yeast to filamentous phase is abolished ultimately when the concentration of undecylenic acid is above 4 mM. Meanwhile, the cell surface is crumpled, and cells display an atrophic appearance under scanning electron microscopy even with low concentration of drug treatment. On the other hand, the drug treatment decreases the transcriptions of hydrolytic enzymes such as secreted aspartic protease, lipase, and phospholipase. Hyphal formation related genes, like HWP1, are significantly reduced in transcriptional level in drug-treated biofilm condition as well. The down-regulated profile of these genes leads to a poorly organized biofilm in undecylenic acid treated environment. PMID:26902505

  6. Fullerene-based inhibitors of HIV-1 protease.

    PubMed

    Strom, T Amanda; Durdagi, Serdar; Ersoz, Suha Salih; Salmas, Ramin Ekhteiari; Supuran, Claudiu T; Barron, Andrew R

    2015-12-01

    A series of Fmoc-Phe(4-aza-C60)-OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4-aza-C60)-OH, is derived from the dipolar addition to C60 of the Fmoc-Nα-protected azido amino acids derived from phenylalanine: Fmoc-Phe(4-aza-C60)-Lys3-OH (1), Fmoc-Phe(4-aza-C60)-Pro-Hyp-Lys-OH (2), and Fmoc-Phe(4-aza-C60)-Hyp-Hyp-Lys-OH (3). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET-based assay to evaluate the enzyme kinetics of HIV-1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc-Phe-Pro-Hyp-Lys-OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C60-based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences.

  7. Interaction of proteases with legume seed inhibitors. Molecular features.

    PubMed

    de Seidl, D S

    1996-12-01

    After having found that raw black beans (Phaseolus vulgaris) were toxic, while the cooked ones constitute the basic diet of the underdeveloped peoples of the world, in the sixties, our research directed by Dr. Jaffé, concentrated mainly around the detection and identification of the heat labile toxic factors in legume seeds. A micromethod for the detection of protease inhibitors (PI) in individual seeds was developed, for the purpose of establishing that the multiple trypsin inhibitors (TI) found in the Cubagua variety were expressions of single seeds and not a mixture of a non homogenous bean lot. Six isoinhibitors were isolated and purified, all of which were "double-headed" and interacted with trypsin (T) and chymotrypsin (CHT) independently and simultaneously, as shown by electrophoresis of their binary and ternary complexes with each and both enzymes. However, their affinity for the enzymes, including elastases, was rather variable, as well as their amino acid composition which consisted of 51 units for inhibitor V, the smallest, and 83 amino acids for inhibitor I, the largest. A low molecular weight protein fraction that inhibited subtilisin (S), but recognized neither T, CHT nor pancreatic elastase was detected in 63 varieties of Phaseolus vulgaris as well as in broad beans (Vicia faba), chick peas (Cicer arietinum), jack beans (Canavalia ensiformis), kidney beans (Vigna aureus), etc., It was absent though, in soybeans (Glycine max), lentils (Lens culinaris), green peas (Pisum sativum), cowpea (Vigna sinensis) and lupine seeds (Lupinus sp). Subtilisin inhibitors (SI) were isolated from black beans, broad beans, chick peas and jack beans. Their Mr is between 8-9KD and they show a rather high stability in the presence of denaturing agents. They are specific toward microbial proteases, in addition to subtilisins, Carlsberg and BPN', they inhibit the alkaline protease from Tritirachium album (Protease K), from Aspergillus oryzae and one isolated from

  8. Proteases of Treponema denticola outer sheath and extracellular vesicles.

    PubMed Central

    Rosen, G; Naor, R; Rahamim, E; Yishai, R; Sela, M N

    1995-01-01

    Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases. PMID:7558307

  9. Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination

    PubMed Central

    Czurda, S.; Smelik, S.; Preuner-Stix, S.; Nogueira, F.

    2015-01-01

    Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays. PMID:26560539

  10. Cleavage and activation of a Toll-like receptor by microbial proteases

    PubMed Central

    de Zoete, Marcel R.; Bouwman, Lieneke I.; Keestra, A. Marijke; van Putten, Jos P. M.

    2011-01-01

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  11. Cleavage and activation of a Toll-like receptor by microbial proteases.

    PubMed

    de Zoete, Marcel R; Bouwman, Lieneke I; Keestra, A Marijke; van Putten, Jos P M

    2011-03-22

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

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

    PubMed

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

    2016-05-01

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

  13. Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability

    PubMed Central

    Stas, Jelle; Nguyen, Nhu H.; Kuyper, Thomas W.; Carleer, Robert; Vangronsveld, Jaco; Colpaert, Jan V.; Kennedy, Peter G.

    2015-01-01

    In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4+ and NO3−) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability. PMID:26682855

  14. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  15. Polyphosphorylated fungal cell wall glycopeptides

    SciTech Connect

    Bonetti, S.J.; Black, B.; Gander, J.E.

    1987-05-01

    Penicillium charlesii secretes a 65 kDa peptidophosphogalactomannan (pPGM) containing 10 phosphodiester residues and 10 galactofuranosyl-containing galactin chains attached to a linear mannan; the polysaccharides is attached to a 3 kDa seryl- and threonyl-rich peptide. The authors have now isolated and partially characterized a form of pPGM released from mycelia of P. charlesii treated at 50/sup 0/C for 15, 30, 60 or 120 min. Two- to 3-fold more pPGM was released by heat treatment than is secreted. Crude pPGM, released by heat, was fractionated on DE-52 and was fractionated into two major fractions on the basis of its difference in negative charge. /sup 1/H-decoupled /sup 13/C NMR spectroscopy of these two fractions provided spectra very similar to that of secreted pPGM previously reported from this laboratory. /sup 1/H-decoupled /sup 31/P NMR showed major signals at 1.47, and 0.22 ppm and minor signals at 1.32, 1.15, 1.00, 0.91 and 0.76 ppm. These signals are upfield from phosphomonoesters and are in the region observed for (6-O-phosphorylcholine)- and (6-O-phosphorylethanolamine)-..cap alpha..-D-mannopyranosyl residues which are 0.22 and 0.90 ppm, respectively. These polymers contain 30 phosphodiester residues per molecule of 70 kDa mass compared with 10 phosphodiesters in secreted pPGM. Acid phosphatase and alkaline protease were the only lytic enzymes released by heat treatment. The evidence suggests that much of the pPGM is derived from cell walls; and that the polysaccharide is highly phosphorylated.

  16. The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia

    PubMed Central

    Gallo, Juan E.; Holder, Jason; Sullivan, Thomas D.; Marty, Amber J.; Carmen, John C.; Chen, Zehua; Ding, Li; Gujja, Sharvari; Magrini, Vincent; Misas, Elizabeth; Mitreva, Makedonka; Priest, Margaret; Saif, Sakina; Whiston, Emily A.; Young, Sarah; Zeng, Qiandong; Goldman, William E.; Mardis, Elaine R.; Taylor, John W.; McEwen, Juan G.; Clay, Oliver K.; Klein, Bruce S.; Cuomo, Christina A.

    2015-01-01

    Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution

  17. Multi-stressor impacts on fungal diversity and ecosystem functions in streams: natural vs. anthropogenic stress.

    PubMed

    Tolkkinen, M; Mykrä, H; Annala, M; Markkola, A M; Vuori, K M; Muotka, T

    2015-03-01

    Biological assemblages are often subjected to multiple stressors emerging from both anthropogenic activities and naturally stressful conditions, and species' responses to simultaneous stressors may differ from those predicted based on the individual effects of each stressor alone. We studied the influence of land-use disturbance (forest drainage) on fungal decomposer assemblages and leaf decomposition rates in naturally harsh (low pH caused by black-shale dominated geology) vs. circumneutral streams. We used pyrosequencing to determine fungal richness and assemblage structure. Decomposition rates did not differ between circumneutral and naturally acidic reference sites. However, the effect of forest drainage on microbial decomposition was more pronounced in the naturally acidic streams than in circumneutral streams. Single-effect responses of fungal assemblages were mainly related to geology. Community similarity was significantly higher in the naturally acidic disturbed sites than in corresponding reference sites, suggesting that land-use disturbance simplifies fungal assemblages in naturally stressful conditions. Naturally acidic streams supported distinct fungal assemblages with many OTUs (operational taxonomic unit) unique to these streams. Our results indicate that fungal assemblages in streams are sensitive to both structural and functional impairment in response to multiple stressors. Anthropogenic degradation of naturally acidic streams may decrease regional fungal diversity and impair ecosystem functions, and these globally occurring environments therefore deserve special attention in conservation planning. PMID:26236864

  18. Multi-stressor impacts on fungal diversity and ecosystem functions in streams: natural vs. anthropogenic stress.

    PubMed

    Tolkkinen, M; Mykrä, H; Annala, M; Markkola, A M; Vuori, K M; Muotka, T

    2015-03-01

    Biological assemblages are often subjected to multiple stressors emerging from both anthropogenic activities and naturally stressful conditions, and species' responses to simultaneous stressors may differ from those predicted based on the individual effects of each stressor alone. We studied the influence of land-use disturbance (forest drainage) on fungal decomposer assemblages and leaf decomposition rates in naturally harsh (low pH caused by black-shale dominated geology) vs. circumneutral streams. We used pyrosequencing to determine fungal richness and assemblage structure. Decomposition rates did not differ between circumneutral and naturally acidic reference sites. However, the effect of forest drainage on microbial decomposition was more pronounced in the naturally acidic streams than in circumneutral streams. Single-effect responses of fungal assemblages were mainly related to geology. Community similarity was significantly higher in the naturally acidic disturbed sites than in corresponding reference sites, suggesting that land-use disturbance simplifies fungal assemblages in naturally stressful conditions. Naturally acidic streams supported distinct fungal assemblages with many OTUs (operational taxonomic unit) unique to these streams. Our results indicate that fungal assemblages in streams are sensitive to both structural and functional impairment in response to multiple stressors. Anthropogenic degradation of naturally acidic streams may decrease regional fungal diversity and impair ecosystem functions, and these globally occurring environments therefore deserve special attention in conservation planning.

  19. Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P.

    PubMed

    Lavey, Nathan P; Coker, Jesse A; Ruben, Eliza A; Duerfeldt, Adam S

    2016-04-22

    Caseinolytic protease P (ClpP) maintains essential roles in bacterial homeostasis. As such, both the inhibition and activation of this enzyme result in bactericidal activity, making ClpP a promising target for antibacterial drug development. Herein, we report the results of a fluorescence-based screen of ∼450 structurally diverse fungal and bacterial secondary metabolites. Sclerotiamide (1), a paraherquamide-related indolinone, was identified as the first non-peptide-based natural product activator of ClpP. Structure-activity relationships arising from the initial screen, preliminary biochemical evaluation of 1, and rationale for the exploitation of this chemotype to develop novel ClpP activators are presented.

  20. Control of the formation of amylases and proteases in the cotyledons of germinating peas.

    PubMed

    Yomo, H; Varner, J E

    1973-04-01

    Protease activity increased in attached cotyledons of germinated peas (Pisum sativum L. cv. Alaska) as the stored proteins declined but did not increase in excised cotyledons incubated for the same length of time. Cotyledons of seeds germinated in the presence of a casein hydrolysate solution developed less protease activity than did those germinated on water. These results suggest that accumulation of amino acids regulates the protease level in the cotyledons of germinating peas.In contrast to protease, alpha- and beta-amylase increased during incubation of excised pea cotyledons. Their increase was inhibited by abscisic acid. Abscisic acid did not inhibit (14)C-leucine incorporation into protein or reduce the respiratory rate in the cotyledons; hence, its effect on amylase formation was not the result of a general inhibition of metabolism. An ether-soluble acid fraction, which would contain any abscisic acid present in the material, inhibited amylase formation more when it was obtained from imbibed seeds than when it was obtained from cotyledons of seeds germinated for 10 days. These and other results suggest that amylase formation in germinating peas is regulated by abscisic acid.

  1. Protease-degradable electrospun fibrous hydrogels

    NASA Astrophysics Data System (ADS)

    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.

  2. The molecular evolution of HIV-1 protease simulated at atomic detail.

    PubMed

    Tiana, G; Broglia, R A

    2009-09-01

    Progress in understanding protein folding allows to simulate, with atomic detail, the evolution of amino-acid sequences folding to a given native conformation. A particularly attractive example is the HIV-1 protease, main target of therapies to fight AIDS, which under drug pressure is able to develop resistance within few months from the starting of therapy. By comparing the results of simulations of the evolution of the protease with the corresponding proteomic data, one can approximately determine the value of the associated evolution pressure under which the enzyme has become and, as a consequence, map out the energy landscape in sequence space of the HIV-1 protease. It is found that there are several families of sequences folding to the native conformations of the enzyme. Each of these families are characterized by different sets of highly conserved ("hot") amino acids which play a critical role in the folding and stability of the protease. There are two main possibilities for the virus to move from one family to a different one: (a) in a single generation, through the concerted mutations of the hot amino acids, a highly unlikely event, (b) through a folding path (if it exists), again a very improbable event. In fact, the number of generations needed by the virus to change stepwise its sequence from one family to another is astronomically large. These results point to the "hot" segments of the protease as promising targets for a nonconventional inhibition strategy, likely not to create resistance.

  3. FungalRV: adhesin prediction and immunoinformatics portal for human fungal pathogens

    PubMed Central

    2011-01-01

    Background The availability of sequence data of human pathogenic fungi generates opportunities to develop Bioinformatics tools and resources for vaccine development towards benefitting at-risk patients. Description We have developed a fungal adhesin predictor and an immunoinformatics database with predicted adhesins. Based on literature search and domain analysis, we prepared a positive dataset comprising adhesin protein sequences from human fungal pathogens Candida albicans, Candida glabrata, Aspergillus fumigatus, Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Blastomyces dermatitidis, Pneumocystis carinii, Pneumocystis jirovecii and Paracoccidioides brasiliensis. The negative dataset consisted of proteins with high probability to function intracellularly. We have used 3945 compositional properties including frequencies of mono, doublet, triplet, and multiplets of amino acids and hydrophobic properties as input features of protein sequences to Support Vector Machine. Best classifiers were identified through an exhaustive search of 588 parameters and meeting the criteria of best Mathews Correlation Coefficient and lowest coefficient of variation among the 3 fold cross validation datasets. The "FungalRV adhesin predictor" was built on three models whose average Mathews Correlation Coefficient was in the range 0.89-0.90 and its coefficient of variation across three fold cross validation datasets in the range 1.2% - 2.74% at threshold score of 0. We obtained an overall MCC value of 0.8702 considering all 8 pathogens, namely, C. albicans, C. glabrata, A. fumigatus, B. dermatitidis, C. immitis, C. posadasii, H. capsulatum and P. brasiliensis thus showing high sensitivity and specificity at a threshold of 0.511. In case of P. brasiliensis the algorithm achieved a sensitivity of 66.67%. A total of 307 fungal adhesins and adhesin like proteins were predicted from the entire proteomes of eight human pathogenic fungal species. The immunoinformatics

  4. Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors.

    PubMed

    Martínez, Dana E; Bartoli, Carlos G; Grbic, Vojislava; Guiamet, Juan J

    2007-01-01

    Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.

  5. Discrimination of fungal infections on grape berries via spectral signatures

    NASA Astrophysics Data System (ADS)

    Molitor, Daniel; Griesser, Michaela; Schütz, Erich; Khuen, Marie-Therese; Schefbeck, Christa; Ronellenfitsch, Franz Kai; Schlerf, Martin; Beyer, Marco; Schoedl-Hummel, Katharina; Anhalt, Ulrike; Forneck, Astrid

    2016-04-01

    The fungal pathogens Botrytis cinerea and Penicillium expansum are causing economic damages on grapevine worldwide. Especially the simultaneous occurrence of both often results in off-flavours highly threatening wine quality. For the classification of grape quality as well as for the determination of targeted enological treatments, the knowledge of the level of fungal attack is of highest interest. However, visual assessment and pathogen discrimination are cost-intensive. Consequently, a pilot laboratory study aimed at (i) detecting differences in spectral signatures between grape berry lots with different levels of infected berries (B. cinerea and/or P. expansum) and (ii) detecting links between spectral signatures and biochemical as well as quantitative molecular markers for fungal attack. To this end, defined percentages (infection levels) of table grape berries were inoculated with fungal spore suspensions. Spectral measurements were taken using a FieldSpec 3 Max spectroradiometer (ASD Inc., Boulder/Colorado, USA) in regular intervals after inoculation. In addition, fungal attack was determined enzymatically) and quantitatively (real-time PCR). In addition, gluconic acid concentrations (as a potential markers for fungal attack) were determined photometrically. Results indicate that based on spectral signatures, a discrimination of P. expansum and B. cinerea infections as well as of different B. cinerea infection levels is possible. Real-time PCR analyses, detecting DNA levels of both fungi, showed yet a low detection level. Whereas the gluconic acid concentrations turned out to be specific for the two fungi tested (B. cinerea vs. P. expansum) and thus may serve as a differentiating biochemical marker. Correlation analyses between spectral measurements and biological data (gluconic acid concentrations, fungi DNA) as well as further common field and laboratory trials are targeted.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

    PubMed Central

    Mourão, Caroline B.F.; Schwartz, Elisabeth F.

    2013-01-01

    The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared. PMID:23771044

  8. Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases.

    PubMed

    Niehaus, F; Gabor, E; Wieland, S; Siegert, P; Maurer, K H; Eck, J

    2011-11-01

    In the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non-redundant subtilase sequence tags with degenerate primers. Furthermore, an activity- as well as a sequence homology-based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full-length protease genes were recovered, sharing only 37-85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin-like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

  9. Protease inhibitors from marine venomous animals and their counterparts in terrestrial venomous animals.

    PubMed

    Mourão, Caroline B F; Schwartz, Elisabeth F

    2013-06-14

    The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared.

  10. Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases

    PubMed Central

    Niehaus, F.; Gabor, E.; Wieland, S.; Siegert, P.; Maurer, K. H.; Eck, J.

    2011-01-01

    Summary In the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non‐redundant subtilase sequence tags with degenerate primers. Furthermore, an activity‐ as well as a sequence homology‐based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full‐length protease genes were recovered, sharing only 37–85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin‐like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications. PMID:21895993

  11. Biochemical properties and potential applications of a solvent-stable protease from the high-yield protease producer Pseudomonas aeruginosa PT121.

    PubMed

    Tang, Xiao-Yu; Wu, Bin; Ying, Han-Jie; He, Bing-Fang

    2010-02-01

    An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60 degrees C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0-11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-L-asparagine (Z-Asp) and L-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.

  12. Cancer Patients and Fungal Infections

    MedlinePlus

    ... mould-related diseases in immunocompromised patients. Journal of Antimicrobial Chemotherapy 2011;66:i5-i14. Ribaud P. Fungal ... al. Clinical Practice Guideline for the Use of Antimicrobial Agents in Neutropenic Patients with Cancer: 2010 Update ...

  13. Evaluation of pre-rigor injection of beef with proteases on cooked meat volatile profile after 1 day and 21 days post-mortem storage.

    PubMed

    Ma, Q L; Hamid, N; Bekhit, A E D; Robertson, J; Law, T F

    2012-12-01

    This research was carried out to determine the effects of pre-rigor injection of beef semimembranosus muscle with nine proteases from plant and microbial sources, on the volatile profile of cooked beef after 1 day and 21 days post-mortem (PM) storage using Solid-phase microextraction gas chromatography mass spectrometry analysis. A total of 23 aldehydes, 5 ketones, 3 furans, 8 nitrogen and sulphur compounds, 4 alkanes, 7 alcohols and 6 terpenes were detected. Eleven volatile compounds characteristic of ginger flavour were detected in zingibain-treated meat. Benzaldehyde significantly increased (p<0.05) only in kiwifruit juice (KJ), fungal 31 protease and Asparagus protease (ASP) treated samples from 1 day to 21 days PM storage. A significant increase (p<0.05) in 3-methylbutanal was observed in KJ, bacterial and fungal protease treated samples at 21 days PM storage. Treatments with bromelain, papain, ASP, actinidin, and KJ (except KJ 21 days) proteases resulted in flavour profiles closer to that of the control beef sample. PMID:22682685

  14. Evaluation of pre-rigor injection of beef with proteases on cooked meat volatile profile after 1 day and 21 days post-mortem storage.

    PubMed

    Ma, Q L; Hamid, N; Bekhit, A E D; Robertson, J; Law, T F

    2012-12-01

    This research was carried out to determine the effects of pre-rigor injection of beef semimembranosus muscle with nine proteases from plant and microbial sources, on the volatile profile of cooked beef after 1 day and 21 days post-mortem (PM) storage using Solid-phase microextraction gas chromatography mass spectrometry analysis. A total of 23 aldehydes, 5 ketones, 3 furans, 8 nitrogen and sulphur compounds, 4 alkanes, 7 alcohols and 6 terpenes were detected. Eleven volatile compounds characteristic of ginger flavour were detected in zingibain-treated meat. Benzaldehyde significantly increased (p<0.05) only in kiwifruit juice (KJ), fungal 31 protease and Asparagus protease (ASP) treated samples from 1 day to 21 days PM storage. A significant increase (p<0.05) in 3-methylbutanal was observed in KJ, bacterial and fungal protease treated samples at 21 days PM storage. Treatments with bromelain, papain, ASP, actinidin, and KJ (except KJ 21 days) proteases resulted in flavour profiles closer to that of the control beef sample.

  15. Mast cell proteases as pharmacological targets.

    PubMed

    Caughey, George H

    2016-05-01

    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

  16. Biosynthesis of Fungal Indole Alkaloids

    PubMed Central

    Xu, Wei; Gavia, Diego J.; Tang, Yi

    2014-01-01

    This review provides a summary of recent research advances in elucidating the biosynthesis of fungal indole alkaloids. Different strategies used to incorporate and derivatize the indole/indoline moieties in various families of fungal indole alkaloids will be discussed, including tryptophan-containing nonribosomal peptides and polyketide-nonribosomal peptide hybrids; and alkaloids derived from other indole building blocks. This review also includes discussion regarding the downstream modifications that generate chemical and structural diversity among indole alkaloids. PMID:25180619

  17. Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora.

    PubMed

    Tunlid, A; Rosén, S; Ek, B; Rask, L

    1994-07-01

    When grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular proteases hydrolysing the chromogenic substrate Azocoll. The protease activity was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PII immobilized P. redivivus in bioassays and hydrolysed proteins of the purified cuticle. The enzyme hydrolysed several protein substrates including casein, bovine serum albumin and gelatin, but not native collagen. Examination of substrate specificity with synthetic peptides showed that PII readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-Gly-Phe-NA). Mono-peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active site histidine residue of the subtilisin family of serine proteases.

  18. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    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

  19. Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease.

    PubMed Central

    Kooi, C; Cox, A; Darling, P; Sokol, P A

    1994-01-01

    Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase. Images PMID:7516312

  20. Traversing the fungal terpenome

    PubMed Central

    Quin, Maureen B.; Flynn, Christopher M.; Schmidt-Dannert, Claudia

    2014-01-01

    Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of trans-humulyl cation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids. PMID:25171145

  1. A cysteine protease isolated from the latex of Ficus microcarpa: purification and biochemical characterization.

    PubMed

    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.

  2. Fragment-Based Screen against HIV Protease

    PubMed Central

    Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

    2009-01-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

  3. Fragment-based screen against HIV protease.

    PubMed

    Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

    2010-03-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

  4. Secretome analysis identifies potential virulence factors of Diplodia corticola, a fungal pathogen involved in cork oak (Quercus suber) decline.

    PubMed

    Fernandes, Isabel; Alves, Artur; Correia, António; Devreese, Bart; Esteves, Ana Cristina

    2014-01-01

    The characterisation of the secretome of phytopathogenic fungi may contribute to elucidate the molecular mechanisms of pathogenesis. This is particularly relevant for Diplodia corticola, a fungal plant pathogen belonging to the family Botryosphaeriaceae, whose genome remains unsequenced. This phytopathogenic fungus is recognised as one of the most important pathogens of cork oak, being related to the decline of cork oak forests in the Iberian Peninsula. Unfortunately, secretome analysis of filamentous fungi is limited by the low protein concentration and by the presence of many interfering substances, such as polysaccharides, which affect the separation and analysis by 1D and 2D gel electrophoresis. We compared six protein extraction protocols concerning their suitability for further application with proteomic workflows. The protocols involving protein precipitation were the most efficient, with emphasis on TCA-acetone protocol, allowing us to identify the most abundant proteins on the secretome of this plant pathogen. Approximately 60% of the spots detected were identified, all corresponding to extracellular proteins. Most proteins identified were carbohydrate degrading enzymes and proteases that may be related to D. corticola pathogenicity. Although the secretome was assessed in a noninfection environment, potential virulence factors such as the putative glucan-β-glucosidase, neuraminidase, and the putative ferulic acid esterase were identified. The data obtained forms a useful basis for a deeper understanding of the pathogenicity and infection biology of D. corticola. Moreover, it will contribute to the development of proteomics studies on other members of the Botryosphaeriaceae.

  5. Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased Influenza A virus cleavage and replication

    PubMed Central

    2012-01-01

    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. PMID:22992220

  6. Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglial crosstalk on peri-synaptic astrocytic endfeet.

    PubMed

    Shavit, Efrat; Michaelson, Daniel M; Chapman, Joab

    2011-11-01

    We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K(+) elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse. PMID:21854391

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

    PubMed

    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 < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  8. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    PubMed

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing. PMID:27386922

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

    PubMed

    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 < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant. PMID:16841690

  10. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    PubMed

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing.

  11. Protease-assisted clarification of black currant juice: synergy with other clarifying agents and effects on the phenol content.

    PubMed

    Landbo, Anne-Katrine R; Pinelo, Manuel; Vikbjerg, Anders F; Let, Mette B; Meyer, Anne S

    2006-09-01

    Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation

  12. Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta

    SciTech Connect

    Roy, Sobhan; Aravind, Penmatsa; Madhurantakam, Chaithanya; Ghosh, Ananta Kumar; Sankaranarayanan, Rajan; Das, Amit Kumar

    2006-07-01

    The crystallization and preliminary X-ray crystallographic analysis of a protease inhibitor from the haemolymph of the Indian tasar silk worm A. mylitta is reported. A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6{sub 3}22, with unit-cell parameters a = b = 60.6, c = 85.1 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 Å.

  13. Whole-cell fungal transformation of precursors into dyes

    PubMed Central

    2010-01-01

    Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important

  14. A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

    PubMed

    Rech, Stefanie S; Heidt, Sven; Requena, Natalia

    2013-09-01

    Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.

  15. Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

    PubMed Central

    Pannkuk, Evan L.; Risch, Thomas S.; Savary, Brett J.

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction. PMID:25785714

  16. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    PubMed

    Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction. PMID:25785714

  17. Expanding Proteome Coverage with Orthogonal-specificity α-Lytic Proteases*

    PubMed Central

    Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.; Ghassemian, Majid; Bandeira, Nuno; Komives, Elizabeth A.

    2014-01-01

    Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin's may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type α-lytic protease (WaLP) and an active site mutant of WaLP, M190A α-lytic protease (MaLP). We assess several relevant factors, including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. When data from separate digestions with trypsin, LysC, WaLP, and MaLP were combined, proteome coverage was increased by 101% relative to that achieved with trypsin digestion alone. To demonstrate how the gained sequence coverage can yield additional post-translational modification information, we show the identification of a number of novel phosphorylation sites in the Schizosaccharomyces pombe proteome and include an illustrative example from the protein MPD2 wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined. PMID:24425750

  18. Proteolytic activity and cysteine protease expression in wheat leaves under severe soil drought and recovery.

    PubMed

    Simova-Stoilova, Lyudmila; Vaseva, Irina; Grigorova, Biliana; Demirevska, Klimentina; Feller, Urs

    2010-01-01

    The involvement of acidic proteases in soil drought response of winter wheat (Triticum aestivum L.) at seedling stage in three cultivars differing in water stress tolerance was studied. Withholding irrigation for seven days resulted in severe drought stress corresponding to 60% leaf water deficit. Stressed plants were recovered by providing optimal water supply for 3 days. Reversible changes in leaf pigment and protein content were registered, being least expressed in the drought-resistant cultivar Katya. Protein loss was inversely related to the increase in total proteolytic activity at pH 5 and in aminopeptidase activity at pH 7. Quantitative differences among the cultivars were established only for azocaseinolytic activity (pH 5). The drought-resistant cultivar (Katya) showed relatively little increase in acid protease activity whereas the highest values of this activity were detected in cultivar Pobeda. In-gel staining for cysteine-activated proteases revealed four to five separate activity bands. The upper band, specifically inhibited by E-64, was raised at severe drought. Transcript abundance of two wheat cysteine proteases -Ta.61026 putative thiol protease, and WCP2 peptidase of papain type was analyzed by RT-PCR. Gene expression of the cysteine proteases under study was suppressed in the drought-tolerant cultivar, while in the less resistant ones it remained unchanged or augmented. The results suggest that lower proteolytic activity and decreased expression of certain cysteine protease genes under water deficit during early developmental stage could be regarded as an indicator for drought resistance of winter wheat cultivars.

  19. Alternaria-derived serine protease activity drives IL-33–mediated asthma exacerbations

    PubMed Central

    Snelgrove, Robert J.; Gregory, Lisa G.; Peiró, Teresa; Akthar, Samia; Campbell, Gaynor A.; Walker, Simone A.; Lloyd, Clare M.

    2014-01-01

    Background The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. Objective We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation. Methods IL-33 levels were quantified in wild-type and ST2−/− mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Results Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Conclusion Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease. PMID:24636086

  20. Anticandidal action of fungal chitosan against Candida albicans.

    PubMed

    Tayel, Ahmed A; Moussa, Shaaban; el-Tras, Wael F; Knittel, Dierk; Opwis, Klaus; Schollmeyer, Eckhard

    2010-11-01

    The anticandidal activity of four fungal chitosan types, produced from Mucor rouxii DSM-1191, against three Candida albicans strains was determined. The most bioactive chitosan type, to inhibit C. albicans growth, had the lowest molecular weight (32 kDa) and the highest deacetylation degree (94%). Water soluble types had stronger anticandidal activity than soluble types in 1% acetic acid solution. Scanning electron micrographs of treated C. albicans with fungal chitosan proved that chitosan principally interact with yeast cell wall, causing severe swelling and asymmetric rough shapes, and subsequent cell wall lyses with the prolonging of exposure time. Fungal chitosan could be recommended for C. albicans control as a powerful and safe alternative to synthetic and chemical fungicides. PMID:20603144

  1. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  2. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  3. Age and Gender Affect the Composition of Fungal Population of the Human Gastrointestinal Tract.

    PubMed

    Strati, Francesco; Di Paola, Monica; Stefanini, Irene; Albanese, Davide; Rizzetto, Lisa; Lionetti, Paolo; Calabrò, Antonio; Jousson, Olivier; Donati, Claudio; Cavalieri, Duccio; De Filippo, Carlotta

    2016-01-01

    The fungal component of the human gut microbiota has been neglected for long time due to the low relative abundance of fungi with respect to bacteria, and only recently few reports have explored its composition and dynamics in health or disease. The application of metagenomics methods to the full understanding of fungal communities is currently limited by the under representation of fungal DNA with respect to the bacterial one, as well as by the limited ability to discriminate passengers from colonizers. Here, we investigated the gut mycobiota of a cohort of healthy subjects in order to reduce the gap of knowledge concerning fungal intestinal communities in the healthy status further screening for phenotypical traits that could reflect fungi adaptation to the host. We studied the fecal fungal populations of 111 healthy subjects by means of cultivation on fungal selective media and by amplicon-based ITS1 metagenomics analysis on a subset of 57 individuals. We then characterized the isolated fungi for their tolerance to gastrointestinal (GI) tract-like challenges and their susceptibility to antifungals. A total of 34 different fungal species were isolated showing several phenotypic characteristics associated with intestinal environment such as tolerance to body temperature (37°C), to acidic and oxidative stress, and to bile salts exposure. We found a high frequency of azoles resistance in fungal isolates, with potential and significant clinical impact. Analyses of fungal communities revealed that the human gut mycobiota differs in function of individuals' life stage in a gender-related fashion. The combination of metagenomics and fungal cultivation allowed an in-depth understanding of the fungal intestinal community structure associated to the healthy status and the commensalism-related traits of isolated fungi. We further discussed comparatively the results of sequencing and cultivation to critically evaluate the application of metagenomics-based approaches to

  4. Age and Gender Affect the Composition of Fungal Population of the Human Gastrointestinal Tract

    PubMed Central

    Strati, Francesco; Di Paola, Monica; Stefanini, Irene; Albanese, Davide; Rizzetto, Lisa; Lionetti, Paolo; Calabrò, Antonio; Jousson, Olivier; Donati, Claudio; Cavalieri, Duccio; De Filippo, Carlotta

    2016-01-01

    The fungal component of the human gut microbiota has been neglected for long time due to the low relative abundance of fungi with respect to bacteria, and only recently few reports have explored its composition and dynamics in health or disease. The application of metagenomics methods to the full understanding of fungal communities is currently limited by the under representation of fungal DNA with respect to the bacterial one, as well as by the limited ability to discriminate passengers from colonizers. Here, we investigated the gut mycobiota of a cohort of healthy subjects in order to reduce the gap of knowledge concerning fungal intestinal communities in the healthy status further screening for phenotypical traits that could reflect fungi adaptation to the host. We studied the fecal fungal populations of 111 healthy subjects by means of cultivation on fungal selective media and by amplicon-based ITS1 metagenomics analysis on a subset of 57 individuals. We then characterized the isolated fungi for their tolerance to gastrointestinal (GI) tract-like challenges and their susceptibility to antifungals. A total of 34 different fungal species were isolated showing several phenotypic characteristics associated with intestinal environment such as tolerance to body temperature (37°C), to acidic and oxidative stress, and to bile salts exposure. We found a high frequency of azoles resistance in fungal isolates, with potential and significant clinical impact. Analyses of fungal communities revealed that the human gut mycobiota differs in function of individuals' life stage in a gender-related fashion. The combination of metagenomics and fungal cultivation allowed an in-depth understanding of the fungal intestinal community structure associated to the healthy status and the commensalism-related traits of isolated fungi. We further discussed comparatively the results of sequencing and cultivation to critically evaluate the application of metagenomics-based approaches to

  5. [Correlation between the synthesis of extracellular proteases and the synthesis of the red pigment prodigiosin in Serratia marcescens].

    PubMed

    Loriia, Zh K; Briukner, B; Egorov, N S

    1977-01-01

    A correlation has been established between synthesis of exocellular protease and synthesis of a red pigment prodigiosine by Serratia marcescens. Chloramphenicol, an inhibitor of protein synthesis, inhibits also synthesis of the pigment. Leucine, an inductor of synthesis of the exocellular protease by Serratia marcescens VI, induces also synthesis of the pigment. A mixture of 18 natural amino acids, asparagine and ammonium ions represses both synthesis of the enzyme and the pigment.

  6. Fungal pathogens of Proteaceae.

    PubMed

    Crous, P W; Summerell, B A; Swart, L; Denman, S; Taylor, J E; Bezuidenhout, C M; Palm, M E; Marincowitz, S; Groenewald, J Z

    2011-12-01

    Species of Leucadendron, Leucospermum and Protea (Proteaceae) are in high demand for the international floriculture market due to their brightly coloured and textured flowers or bracts. Fungal pathogens, however, create a serious problem in cultivating flawless blooms. The aim of the present study was to characterise several of these pathogens using morphology, culture characteristics, and DNA sequence data of the rRNA-ITS and LSU genes. In some cases additional genes such as TEF 1-α and CHS were also sequenced. Based on the results of this study, several novel species and genera are described. Brunneosphaerella leaf blight is shown to be caused by three species, namely B. jonkershoekensis on Protea repens, B. nitidae sp. nov. on Protea nitida and B. protearum on a wide host range of Protea spp. (South Africa). Coniothyrium-like species associated with Coniothyrium leaf spot are allocated to other genera, namely Curreya grandicipis on Protea grandiceps, and Microsphaeropsis proteae on P. nitida (South Africa). Diaporthe leucospermi is described on Leucospermum sp. (Australia), and Diplodina microsperma newly reported on Protea sp. (New Zealand). Pyrenophora blight is caused by a novel species, Pyrenophora leucospermi, and not Drechslera biseptata or D. dematoidea as previously reported. Fusicladium proteae is described on Protea sp. (South Africa), Pestalotiopsis protearum on Leucospermum cuneiforme (Zimbabwe), Ramularia vizellae and R. stellenboschensis on Protea spp. (South Africa), and Teratosphaeria capensis on Protea spp. (Portugal, South Africa). Aureobasidium leaf spot is shown to be caused by two species, namely A. proteae comb. nov. on Protea spp. (South Africa), and A. leucospermi sp. nov. on Leucospermum spp. (Indonesia, Portugal, South Africa). Novel genera and species elucidated in this study include Gordonomyces mucovaginatus and Pseudopassalora gouriqua (hyphomycetes), and Xenoconiothyrium catenata (coelomycete), all on Protea spp. (South Africa).

  7. The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

    SciTech Connect

    Bandaranayake, Rajintha M.; Kolli, Madhavi; King, Nancy M.; Nalivaika, Ellen A.; Heroux, Annie; Kakizawa, Junko; Sugiura, Wataru; Schiffer, Celia A.

    2010-09-08

    The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01{_}AE (AE) strain is seen principally in Southeast Asia. AE protease differs by {approx}10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

  8. Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

    PubMed

    Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

    2006-01-01

    An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

  9. Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

    PubMed

    Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

    2006-01-01

    An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA. PMID:16394504

  10. Fungal Community Assembly in the Amazonian Dark Earth.

    PubMed

    Lucheta, Adriano Reis; Cannavan, Fabiana de Souza; Roesch, Luiz Fernando Wurdig; Tsai, Siu Mui; Kuramae, Eiko Eurya

    2016-05-01

    Here, we compare the fungal community composition and diversity in Amazonian Dark Earth (ADE) and the respective non-anthropogenic origin adjacent (ADJ) soils from four different sites in Brazilian Central Amazon using pyrosequencing of 18S ribosomal RNA (rRNA) gene. Fungal community composition in ADE soils were more similar to each other than their ADJ soils, except for only one site. Phosphorus and aluminum saturation were the main soil chemical factors contributing to ADE and ADJ fungal community dissimilarities. Differences in fungal richness were not observed between ADE and ADJ soil pairs regarding to the most sites. In general, the most dominant subphyla present in the soils were Pezizomycotina, Agaricomycotina, and Mortierellomycotina. The most abundant operational taxonomic units (OTUs) in ADE showed similarities with the entomopathogenic fungus Cordyceps confragosa and the saprobes Fomitopsis pinicola, Acremonium vitellinum, and Mortierellaceae sp., whereas OTUs similar to Aspergillus niger, Lithothelium septemseptatum, Heliocephala gracillis, and Pestalosphaeria sp. were more abundant in ADJ soils. Differences in fungal community composition were associated to soil chemical factors in ADE (P, Ca, Zn, Mg, organic matter, sum of bases, and base saturation) and ADJ (Al, potential acidity, Al saturation, B, and Fe) soils. These results contribute to a deeper view of the fungi communities in ADE and open new perspectives for entomopathogenic fungi studies. PMID:26585119

  11. Fungal degradation of coal as a pretreatment for methane production

    USGS Publications Warehouse

    Haider, Rizwan; Ghauri, Muhammad A.; SanFilipo, John R.; Jones, Elizabeth J.; Orem, William H.; Tatu, Calin A.; Akhtar, Kalsoom; Akhtar, Nasrin

    2013-01-01

    Coal conversion technologies can help in taking advantage of huge low rank coal reserves by converting those into alternative fuels like methane. In this regard, fungal degradation of coal can serve as a pretreatment step in order to make coal a suitable substrate for biological beneficiation. A fungal isolate MW1, identified as Penicillium chrysogenum on the basis of fungal ITS sequences, was isolated from a core sample of coal, taken from a well drilled by the US. Geological Survey in Montana, USA. The low rank coal samples, from major coal fields of Pakistan, were treated with MW1 for 7 days in the presence of 0.1% ammonium sulfate as nitrogen source and 0.1% glucose as a supplemental carbon source. Liquid extracts were analyzed through Excitation–Emission Matrix Spectroscopy (EEMS) to obtain qualitative estimates of solubilized coal; these analyses indicated the release of complex organic functionalities. In addition, GC–MS analysis of these extracts confirmed the presence of single ring aromatics, polyaromatic hydrocarbons (PAHs), aromatic nitrogen compounds and aliphatics. Subsequently, the released organics were subjected to a bioassay for the generation of methane which conferred the potential application of fungal degradation as pretreatment. Additionally, fungal-mediated degradation was also prospected for extracting some other chemical entities like humic acids from brown coals with high huminite content especially from Thar, the largest lignite reserve of Pakistan.

  12. Fungal Community Assembly in the Amazonian Dark Earth.

    PubMed

    Lucheta, Adriano Reis; Cannavan, Fabiana de Souza; Roesch, Luiz Fernando Wurdig; Tsai, Siu Mui; Kuramae, Eiko Eurya

    2016-05-01

    Here, we compare the fungal community composition and diversity in Amazonian Dark Earth (ADE) and the respective non-anthropogenic origin adjacent (ADJ) soils from four different sites in Brazilian Central Amazon using pyrosequencing of 18S ribosomal RNA (rRNA) gene. Fungal community composition in ADE soils were more similar to each other than their ADJ soils, except for only one site. Phosphorus and aluminum saturation were the main soil chemical factors contributing to ADE and ADJ fungal community dissimilarities. Differences in fungal richness were not observed between ADE and ADJ soil pairs regarding to the most sites. In general, the most dominant subphyla present in the soils were Pezizomycotina, Agaricomycotina, and Mortierellomycotina. The most abundant operational taxonomic units (OTUs) in ADE showed similarities with the entomopathogenic fungus Cordyceps confragosa and the saprobes Fomitopsis pinicola, Acremonium vitellinum, and Mortierellaceae sp., whereas OTUs similar to Aspergillus niger, Lithothelium septemseptatum, Heliocephala gracillis, and Pestalosphaeria sp. were more abundant in ADJ soils. Differences in fungal community composition were associated to soil chemical factors in ADE (P, Ca, Zn, Mg, organic matter, sum of bases, and base saturation) and ADJ (Al, potential acidity, Al saturation, B, and Fe) soils. These results contribute to a deeper view of the fungi communities in ADE and open new perspectives for entomopathogenic fungi studies.

  13. Proteases in cellular slime mold development: evidence for their involvement.

    PubMed Central

    Fong, D; Bonner, J T

    1979-01-01

    Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation. Images PMID:293735

  14. Stimulation of the herpes simplex virus type I protease by antichaeotrophic salts.

    PubMed

    Yamanaka, G; DiIanni, C L; O'Boyle, D R; Stevens, J; Weinheimer, S P; Deckman, I C; Matusick-Kumar, L; Colonno, R J

    1995-12-15

    The herpes simplex virus type 1 protease is expressed as an 80,000-dalton polypeptide, encoded within the 635-amino acid open reading frame of the UL26 gene. The two known protein substrates for this enzyme are the protease itself and the capsid assembly protein ICP35 (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). In this report we describe the use of a rapid and quantitative assay for characterizing the protease. The assay uses a glutathione S-transferase fusion protein containing the COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The protease consists of N0, the NH2-terminal 247 amino acid catalytic domain of the UL26 gene product, also expressed as a GST fusion protein. Upon cleavage with N0, a single 25-mer peptide is released from GST-56, which is soluble in trichloroacetic acid. Using this assay, the protease displayed a pH optimum between 7 and 9 but most importantly had an absolute requirement for high concentrations of an antichaeotrophic agent. Strong salting out salts such as Na2SO4 and KPO4 (> or = 1 M) stimulated activity, whereas NaCl and KCl had no effect. The degree of stimulation by 1.25 M Na2SO4 and KPO4 were 100-150- and 200-300-fold, respectively. Using the fluorescent probe 1-anilino-8-naphthalene sulfonate, the protease was shown to bind the dye in the presence of 1.25 M Na2SO4 or KPO4, but not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site because a high affinity cleavage site peptide, but not a control peptide, could displace the dye. In addition to cleaving GST-56, the herpes simplex virus type I protease also cleaved the purified 56-mer peptide. Circular dichroism and NMR spectroscopy showed the peptide to be primarily random coil under physiological conditions, suggesting that antichaeotrophic agents affect the conformation of the substrate as well as the protease.

  15. A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization.

    PubMed

    Khan, Sadaf; Verma, Giti; Sharma, Samir

    2010-07-15

    Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.

  16. Intracellular Action of a Secreted Peptide Required for Fungal Virulence.

    PubMed

    Homer, Christina M; Summers, Diana K; Goranov, Alexi I; Clarke, Starlynn C; Wiesner, Darin L; Diedrich, Jolene K; Moresco, James J; Toffaletti, Dena; Upadhya, Rajendra; Caradonna, Ippolito; Petnic, Sarah; Pessino, Veronica; Cuomo, Christina A; Lodge, Jennifer K; Perfect, John; Yates, John R; Nielsen, Kirsten; Craik, Charles S; Madhani, Hiten D

    2016-06-01

    Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence. PMID:27212659

  17. Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease.

    PubMed Central

    Meek, T D; Dayton, B D; Metcalf, B W; Dreyer, G B; Strickler, J E; Gorniak, J G; Rosenberg, M; Moore, M L; Magaard, V W; Debouck, C

    1989-01-01

    Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease. Images PMID:2648384

  18. Evidence of protease in the saliva of the butterfly Heliconius melpomene (L.) (Nymphalidae, Lepidoptera).

    PubMed

    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.

  19. Vanadium inhibition of serine and cysteine proteases.

    PubMed

    Guerrieri, N; Cerletti, P; De Vincentiis, M; Salvati, A; Scippa, S

    1999-03-01

    A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.

  20. Bioinformatics of proteases in the MEROPS database.

    PubMed

    Barrett, Alan J

    2004-05-01

    Proteolytic enzymes represent approximately approximately 2% of the total number of proteins present in all types of organisms. Many of these enzymes are of medical importance, and those that are of potential interest as drug targets can be divided into the endogenous enzymes encoded in the human genome, and the exogenous proteases encoded in the genomes of disease-causing organisms. There are also naturally occurring inhibitors of proteases, some of which have pharmaceutical relevance. The MEROPS database provides a rich source of information on proteases and their inhibitors. Storage and retrieval of this information is facilitated by the use of a hierarchical classification system (which was pioneered by the compilers of the database) in which homologous proteases and their inhibitors are divided into clans and families. PMID:15216937

  1. Primary immunodeficiencies underlying fungal infections

    PubMed Central

    Lanternier, Fanny; Cypowyj, Sophie; Picard, Capucine; Bustamante, Jacinta; Lortholary, Olivier; Casanova, Jean-Laurent; Puel, Anne

    2014-01-01

    Purpose of review We review the primary immunodeficiencies underlying an increasing variety of superficial and invasive fungal infections. We also stress that the occurrence of such fungal infections should lead physicians to search for the corresponding single-gene inborn errors of immunity. Finally, we suggest that other fungal infections may also result from hitherto unknown inborn errors of immunity, at least in some patients with no known risk factors. Recent findings An increasing number of primary immunodeficiencies are being shown to underlie fungal infectious diseases in children and young adults. Inborn errors of the phagocyte NADPH oxidase complex (chronic granulomatous disease), severe congenital neutropenia and leukocyte adhesion deficiency type I confer a predisposition to invasive aspergillosis and candidiasis. More rarely, inborn errors of IFN-γ immunity underlie endemic mycoses. Inborn errors of IL-17 immunity have recently been shown to underlie chronic mucocutaneous candidiasis, whereas inborn errors of CARD9 immunity underlie deep dermatophytosis and invasive candidiasis. Summary Chronic mucocutaneous candidiasis, invasive candidiasis, invasive aspergillosis, deep dermatophytosis, pneumocystosis, and endemic mycoses can all be caused by primary immunodeficiencies. Each type of infection is highly suggestive of a specific type of primary immunodeficiency. In the absence of overt risk factors, single-gene inborn errors of immunity should be sought in children and young adults with these and other fungal diseases. PMID:24240293

  2. Electrically sensing protease activity with nanopores

    NASA Astrophysics Data System (ADS)

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  3. [Suppression of three soil-borne diseases of cucumber by a rhizosphere fungal strain].

    PubMed

    Lyu, Heng; Niu, Yong-chun; Deng, Hui; Lin, Xiao-min; Jin, Chun-li

    2015-12-01

    To understand the effect of rhizosphere fungi on soil-borne diseases of cucumber, 16 fungal, strains from rhizosphere soil were investigated for the antagonistic activity to three soilborne pathogenic fungi with dual culture method and for suppression of cucumber diseases caused by the pathogens in pot experiments. Four strains showed antagonism to one or more pathogenic fungi tested. The strain JCL143, identified as Aspergillus terreus, showed strong antagonistic activity to the three pathogenic fungi Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani and Sclerotinia sclerotiorum. In greenhouse pot experiments, inoculation with strain JCL143 provided 74% or more of relative control effect to all the three diseases of cucumber seedling caused by the above three pathogenic fungi, and provided 85% or more of relative control effect to Rhizoctonia root rot and Sclerotinia root and stem rot in pot experiment with non-sterilized substrate. In pot experiment with natural soil as substrate, inoculation with strain JCL143 provided average 84.1% of relative control effect to Fusarium wilt of cucumber at vine elongation stage. The fermentation broth of strain JCL143 showed inhibitory effect in different degrees on the colonial growth of the three pathogenic fungi tested, and reached 63.3% of inhibitory rate of colonial growth to S. sclerotiorum. The inhibitory activity of the fermentation broth decreased with increasing treatment temperature, was liable to decrease to alkaline pH than acid pH, and stable to protease treatment. The results indicated that A. terreus is an important factor in suppression of plant soil-borne diseases, and strain JCL143 with stable disease suppression is potential in biocontrol application. PMID:27112016

  4. HIV Protease Inhibitors and Obesity

    PubMed Central

    Anuurad, Erdembileg; Bremer, Andrew; Berglund, Lars

    2011-01-01

    Purpose of review To review the current scientific literature and recent clinical trials on HIV protease inhibitors (PIs) and their potential role in the pathogenesis of lipodystrophy and metabolic disorders. Recent findings HIV PI treatment may affect the normal stimulatory effect of insulin on glucose and fat storage. Further, chronic inflammation from HIV infection and PI treatment trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. This process leads to a pathologic cycle of lipodystrophy and lipotoxicity, a pro-atherogenic lipid profile, and a clinical phenotype of increased central body fat distribution similar to the metabolic syndrome. Summary PIs are a key component of antiretroviral therapy and have dramatically improved the life expectancy of HIV-infected individuals. However, they are also associated with abnormalities in glucose/lipid metabolism and body fat distribution. Further studies are needed to better define the pathogenesis of PI-associated metabolic and body fat changes and their potential treatment. PMID:20717021

  5. ADAM Proteases and Gastrointestinal Function.

    PubMed

    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. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  7. ADAM Proteases and Gastrointestinal Function

    PubMed Central

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

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs