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Sample records for acid fungal protease

  1. Acid protease production in fungal root endophytes.

    PubMed

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

    2015-01-01

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

  2. Impact of an acid fungal protease in high gravity fermentation for ethanol production using Indian sorghum as a feedstock.

    PubMed

    Gohel, V; Duan, G; Maisuria, V B

    2013-01-01

    This study evaluated the conventional jet cooking liquefaction process followed by simultaneous saccharification and fermentation (SSF) at 30% and 35% dry solids (DS) concentration of Indian sorghum feedstock for ethanol production, with addition of acid fungal protease or urea. To evaluate the efficacy of thermostable α-amylase in liquefaction at 30% and 35% DS concentration of Indian sorghum, liquefact solubility, higher dextrins, and fermentable sugars were analyzed at the end of the process. The liquefact was further subjected to SSF using yeast. In comparison with urea, addition of an acid fungal protease during SSF process was observed to accelerate yeast growth (μ), substrate consumption (Q(s)), ultimately ethanol yield based on substrate (Y(p/s)) and ethanol productivity based on fermentation time (Q(p)). The fermentation efficiency and ethanol recovery were determined for both concentrations of Indian sorghum and found to be increased with use of acid fungal protease in SSF process.

  3. 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

  4. 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.

  5. 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.

  6. Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum

    PubMed Central

    Kautto, Liisa; Nevalainen, Helena

    2017-01-01

    Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases. PMID:28060882

  7. Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

    PubMed

    Han, Zhiping; Kautto, Liisa; Nevalainen, Helena

    2017-01-01

    Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

  8. 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.

  9. 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

  10. Homology among acid proteases: comparison of crystal structures at 3A resolution of acid proteases from Rhizopus chinensis and Endothia parasitica.

    PubMed Central

    Subramanian, E; Swan, I D; Liu, M; Davies, D R; Jenkins, J A; Tickle, I J; Blundell, T L

    1977-01-01

    The molecular structures of two fungal acid proteases at 3 A resolution have been compared, and found to have similar secondary and tertiary folding. These enzymes are bilobal and have a pronounced cleft between the two lobes. This cleft has been identified as the active site region from inhibitor binding studies. The results of the comparison are discussed in terms of homology among the acid proteases in general. Images PMID:322132

  11. 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.

  12. Fungal fermentation of whey incorporated with certain supplements for the production of proteases.

    PubMed

    Ashour, S A; el-Shora, H M; Metwally, M; Habib, S A

    1996-01-01

    The pattern and the extent of formation of proteases and secretion varied with the fungus, age and/or the nature of the co-supplement. Addition of yeast extract induced the best yield of proteases from both Aspergillus niger and A. terreus. Proteases from A. niger were highly induced by glutamic acid, alanine or albumin, with minor differences, whereas gelatin, peptone, aspartic acid, casein or acetamide stimulated the accumulation of proteases in the culture medium of A. terreus. Galactose stimulated the yield of the enzyme particularly with A. terreus while most C-supplements inhibited such processes, more prominently in the presence of cellulose or starch. Incorporation of nicotinic acid and wheat bran initiated the maximum productivity of proteases from A. niger and A. terreus, respectively. Co+2 and Cu+2 highly stimulated the output of proteases from A. niger and A. terreus, respectively. Co+2 and Ca+2 stimulated the enzyme activity of alkaline and acid proteases from A. terreus and acid proteases from A. niger. The other six cations and DTT inhibited variably the three proteases particularly alkaline proteases from A. terreus indicating that proteases from various sources even from closely related species showed different properties.

  13. 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.

  14. Multiplexed detection of fungal nucleic acid signatures.

    PubMed

    Diaz, Mara R; Dunbar, Sherry A; Jacobson, James W

    2008-04-01

    Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high-throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies.

  15. Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese.

    PubMed

    Ozturkoglu-Budak, Sebnem; Wiebenga, Ad; Bron, Peter A; de Vries, Ronald P

    2016-11-21

    We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable

  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. 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.

  18. 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.

  19. Effect of the fungal extracellular protease EPg222 on texture of whole pieces of pork loin.

    PubMed

    Benito, María J; Rodríguez, Mar; Acosta, Raquel; Córdoba, Juan J

    2003-10-01

    Proteolytic activity of the fungal protease EPg222 and its effect on texture has been investigated in a meat model system based on sterile pieces of pork loins incubated for 32 days. SDS-PAGE analysis of treated samples showed intense hydrolysis of the myofibrillar proteins H-meromyosin, T-troponin, tropomyosin and the proteins bands of 98, 89, 48, 39, 37 and 28 kDa after 17 days of incubation. Proteolytic activity of the enzyme led to a higher accumulation of NPN in treated than untreated samples. Microstructural analysis of muscle fibres showed loss of muscle fibre structure only in treated batch. The texture profile analysis reveals lower values in hardness, gumminess and chewiness in treated than control batch. This effect may be of great interest in dry-cured meat products to counterbalance the increase of hardness reported in these products as consequence of protein denaturation.

  20. Fungal Effector Protein AVR2 Targets Diversifying Defense-Related Cys Proteases of Tomato[W

    PubMed Central

    Shabab, Mohammed; Shindo, Takayuki; Gu, Christian; Kaschani, Farnusch; Pansuriya, Twinkal; Chintha, Raju; Harzen, Anne; Colby, Tom; Kamoun, Sophien; van der Hoorn, Renier A.L.

    2008-01-01

    The interaction between the fungal pathogen Cladosporium fulvum and its host tomato (Solanum lycopersicum) is an ideal model to study suppression of extracellular host defenses by pathogens. Secretion of protease inhibitor AVR2 by C. fulvum during infection suggests that tomato papain-like cysteine proteases (PLCPs) are part of the tomato defense response. We show that the tomato apoplast contains a remarkable diversity of PLCP activities with seven PLCPs that fall into four different subfamilies. Of these PLCPs, transcription of only PIP1 and RCR3 is induced by treatment with benzothiadiazole, which triggers the salicylic acid–regulated defense pathway. Sequencing of PLCP alleles of tomato relatives revealed that only PIP1 and RCR3 are under strong diversifying selection, resulting in variant residues around the substrate binding groove. The doubled number of variant residues in RCR3 suggests that RCR3 is under additional adaptive selection, probably to prevent autoimmune responses. AVR2 selectively inhibits only PIP1 and RCR3, and one of the naturally occurring variant residues in RCR3 affects AVR2 inhibition. The higher accumulation of PIP1 protein levels compared with RCR3 indicates that PIP1 might be the real virulence target of AVR2 and that RCR3 acts as a decoy for AVR2 perception in plants carrying the Cf-2 resistance gene. PMID:18451324

  1. 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.

  2. Proteases.

    PubMed

    Barrett, A J

    2001-05-01

    The processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly-synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein--e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

  3. Effect of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage "salchichón" ripened with commercial starter cultures.

    PubMed

    Benito, María J; Rodríguez, Mar; Martín, Alberto; Aranda, Emilio; Córdoba, Juan J

    2004-07-01

    The effect of the addition of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage "salchichón" ripened with commercial starter cultures was investigated. Sausages were prepared with purified EPg222 and Staphylococcus carnosus, Staphylococcus xylosus, and Lactobacillus sakei as starter cultures, ripened for 145 days and compared with a control batch only inoculated with the starter cultures. Dry fermented sausages ripened with EPg222 and starter cultures showed higher amount of NPN and volatile compounds derived from amino acid catabolism, than control ripened only with starter cultures. Several branched aldehydes, acids and alcohols such as 2- and 3-methylbutanoic acid and 2-methylpropanol were detected only in enzyme treated samples. Sensory analysis reflected higher values for aroma intensity of sausages treated with EPg222 and lower values of hardness than control. The effect of EPg222 may be of great interest to improve sensory characteristics of dry fermented sausages ripened with starter cultures.

  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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Gibberellic Acid-Induced Synthesis of Protease by Isolated Aleurone Layers of Barley 1

    PubMed Central

    Jacobsen, John V.; Varner, J. E.

    1967-01-01

    The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases. PMID:16656695

  12. 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.

  13. Functional Diversity in Fungal Fatty Acid Synthesis

    PubMed Central

    Blacklock, Brenda J.; Scheffler, Brian E.; Shepard, Michael R.; Jayasuriya, Naomi; Minto, Robert E.

    2010-01-01

    Acetylenic specialized metabolites containing one or more carbon-carbon triple bonds are widespread, being found in fungi, vascular and lower plants, marine sponges and algae, and insects. Plants, moss, and most recently, insects, have been shown to employ an energetically difficult, sequential dehydrogenation mechanism for acetylenic bond formation. Here, we describe the cloning and heterologous expression in yeast of a linoleoyl 12-desaturase (acetylenase) and a bifunctional desaturase with Δ12-/Δ14-regiospecificity from the Pacific golden chanterelle. The acetylenase gene, which is the first identified from a fungus, is phylogenetically distinct from known plant and fungal desaturases. Together, the bifunctional desaturase and the acetylenase provide the enzymatic activities required to drive oleate through linoleate to crepenynate and the conjugated enyne (14Z)-dehydrocrepenynate, the branchpoint precursors to a major class of acetylenic natural products. PMID:20606235

  14. Highly Efficient Gluten Degradation by Lactobacilli and Fungal Proteases during Food Processing: New Perspectives for Celiac Disease▿

    PubMed Central

    Rizzello, Carlo G.; De Angelis, Maria; Di Cagno, Raffaella; Camarca, Alessandra; Silano, Marco; Losito, Ilario; De Vincenzi, Massimo; De Bari, Maria D.; Palmisano, Francesco; Maurano, Francesco; Gianfrani, Carmen; Gobbetti, Marco

    2007-01-01

    Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity

  15. 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-...

  16. 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.

  17. 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

  18. Conservation of cysteine residues in fungal histidine acid phytases.

    PubMed

    Mullaney, Edward J; Ullah, Abul H J

    2005-03-11

    Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

  19. Aspartic acid protease from Botrytis cinerea removes haze-forming proteins during white winemaking.

    PubMed

    Van Sluyter, Steven C; Warnock, Nicholas I; Schmidt, Simon; Anderson, Peter; van Kan, Jan A L; Bacic, Antony; Waters, Elizabeth J

    2013-10-09

    White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the enzymatic removal of haze-forming grape pathogenesis-related proteins using added proteases. The major problem with this approach is that grape pathogenesis-related proteins are highly protease resistant unless they are heat denatured in combination with enzymatic treatment. This paper demonstrates that the protease BcAP8, from the grape fungal pathogen Botrytis cinerea , is capable of degrading chitinase, a major class of haze-forming proteins, without heat denaturation. Because BcAP8 effectively removes haze-forming proteins under normal winemaking conditions, it could potentially benefit winemakers by reducing bentonite requirements.

  20. 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.

  1. 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.

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

    PubMed Central

    Walasek, Paula; Honek, John F

    2005-01-01

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

  3. 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

    2012-12-17

    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.

  4. 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

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

    PubMed

    Monnerie, Hubert; Le Roux, Peter D

    2008-09-01

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

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

    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

  7. Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe.

    PubMed

    Greco, Maria Raffaella; Antelmi, Ester; Busco, Giovanni; Guerra, Lorenzo; Rubino, Rosa; Casavola, Valeria; Reshkin, Stephan Joel; Cardone, Rosa Angela

    2014-02-01

    Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1‑dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

  8. Expression of a Serine Protease Gene prC Is Up-Regulated by Oxidative Stress in the Fungus Clonostachys rosea: Implications for Fungal Survival

    PubMed Central

    Liu, Wen-Jing; Zhou, Wei; Tao, Nan; Tu, Hui-Hui; Huang, Xiao-Wei; Yang, Jin-Kui; Zhang, Ke-Qin

    2010-01-01

    Background Soil fungi face a variety of environmental stresses such as UV light, high temperature, and heavy metals. Adaptation of gene expression through transcriptional regulation is a key mechanism in fungal response to environmental stress. In Saccharomyces cerevisiae, the transcription factors Msn2/4 induce stress-mediated gene expression by binding to the stress response element. Previous studies have demonstrated that the expression of extracellular proteases is up-regulated in response to heat shock in fungi. However, the physiological significance of regulation of these extracellular proteases by heat shock remains unclear. The nematophagous fungus Clonostachys rosea can secret an extracellular serine protease PrC during the infection of nematodes. Since the promoter of prC has three copies of the stress response element, we investigated the effect of environmental stress on the expression of prC. Methodology/Principal Findings Our results demonstrated that the expression of prC was up-regulated by oxidants (H2O2 or menadione) and heat shock, most likely through the stress response element. After oxidant treatment or heat shock, the germination of conidia in the wild type strain was significantly higher than that in the prC mutant strain in the presence of nematode cuticle. Interestingly, the addition of nematode cuticle significantly attenuated the production of reactive oxygen species (ROS) induced by oxidants and heat shock in the wild type strain, but not in prC mutant strain. Moreover, low molecule weight (<3 kD) degradation products of nematode cuticle suppressed the inhibitory effect of conidial germination induced by oxidants and heat shock. Conclusions/Significance These results indicate that PrC plays a protective role in oxidative stress in C. rosea. PrC degrades the nematode cuticle to produce degradation products, which in turn offer a protective effect against oxidative stress by scavenging ROS. Our study reveals a novel strategy for fungi to

  9. The effects of bioprocess parameters on extracellular proteases in a recombinant Aspergillus niger B1-D.

    PubMed

    Li, Qiang; Harvey, Linda M; McNeil, Brian

    2008-02-01

    Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45 degrees C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35 degrees C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.

  10. 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.

  11. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius.

    PubMed

    Lin, X; Tang, J

    1990-01-25

    A thermostable, acid proteolytic activity has been found to be associated with the cells and in the culture medium of Sulfolobus acidocaldarius, an archaebacterium. This acid protease, which has been named thermopsin, was purified to homogeneity from the culture medium by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, monoQ (fast protein liquid chromatography), and gel filtration (high pressure liquid chromatography). The purified thermopsin produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteolytic activity was associated with the band. Thermopsin is a single-chain protein as indicated by gel electrophoresis and by a single NH2-terminal sequence. It has maximal proteolytic activity at pH 2 and 90 degrees C. A genomic library of S. acidocaldarius was prepared and screened by an oligonucleotide probe designed from the NH2-terminal sequence of thermopsin. Five positive clones were isolated. From these clones the thermopsin gene was mapped and sequenced. The nucleotide sequence showed that the thermopsin structure is encoded in 1020 bases. In the deduced protein sequence, there are 41 amino acid residues (including the initiation Met) preceding the NH2-terminal position of thermopsin. Most of these residues appear to be characteristic of a leader sequence. However, the presence in this region of a short pro sequence cannot be ruled out. Thermopsin contains a single cysteine at residue 237 that is not essential for activity (Fusek, M., Lin, X.-L., Tang, J. (1990) J. Biol. Chem. 265, 1496-1501. Thermopsin has no apparent sequence similarity to aspartic proteases of the pepsin family nor to pepstatin-insensitive acid protease (Maita, T., Nagata, S., Matsuda, G., Murata, S., Oda, K., Murao, S., and Tsura, D. (1984) J. Biochem. 95, 465-475) and thus may represent a new class of acid proteases. Also absent is the characteristic active site aspartyl sequence

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

    PubMed Central

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

    2012-01-01

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

  13. 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...

  14. Yeast extracellular proteases.

    PubMed

    Ogrydziak, D M

    1993-01-01

    Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

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

    SciTech Connect

    Baath, E.; Lundgren, B.; Soederstroem, B.

    1984-01-01

    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/sup -1/ each year for 6 years, yet no differences were found between the control treatment and an application of 50 kg ha/sup -1/. The abundance of Penicillium spinulosum and Oidiodendron 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. 15 references, 4 tables.

  16. Fungal variegatic acid and extracellular polysaccharides promote the site-specific generation of reactive oxygen species.

    PubMed

    Zhu, Yuan; Mahaney, James; Jellison, Jody; Cao, Jinzhen; Gressler, Julia; Hoffmeister, Dirk; Goodell, Barry

    2017-03-01

    This study aims to clarify the role of variegatic acid (VA) in fungal attack by Serpula lacrymans, and also the generation and scavenging of reactive oxygen species (ROS) by the fungus. VA promotes a mediated Fenton reaction to generated ROS after oxalate solubilizes oxidized forms of iron. The fungal extracellular matrix (ECM) β-glucan scavenged ROS, and we propose this as a mechanism to protect the fungal hyphae while ROS generation is promoted to deconstruct the lignocellulose cell wall. A relatively high pH (4.4) also favored Fe(III) transfer from oxalate to VA as opposed to a lower pH (2.2) conditions, suggesting a pH-dependent Fe(III) transfer to VA employed by S. lacrymans. This permits ROS generation within the higher pH of the cell wall, while limiting ROS production near the fungal hyphae, while β-glucan from the fungal ECM scavenges ROS in the more acidic environments surrounding the fungal hyphae.

  17. Expanding the product portfolio of fungal type I fatty acid synthases.

    PubMed

    Zhu, Zhiwei; Zhou, Yongjin J; Krivoruchko, Anastasia; Grininger, Martin; Zhao, Zongbao K; Nielsen, Jens

    2017-04-01

    Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.

  18. 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.

  19. 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

  20. Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

    PubMed

    Oliveira, Danielle M P; Ramos, Isabela B; Reis, Flavia C G; Lima, Ana P C A; Machado, Ednildo A

    2008-05-01

    In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

  1. Synthesis of (R)- and (S)-10,16-dihydroxyhexadecanoic acid: cutin stereochemistry and fungal activation.

    PubMed

    Ahmed, Aqeel; Crawford, Terry; Gould, Stephanie; Ha, Y S; Hollrah, Monica; Noor-E-Ain, Farhana; Dickman, Martin B; Dussault, Patrick H

    2003-05-01

    The first asymmetric syntheses of the cutin monomers (R)- and (S)-10,16-dihydroxyhexadecanoic acid (10,16-DHPA) and confirmation of (S)(+)-absolute configuration for 10,16-DHPA derived from tomato are reported. The individual DHPA stereoisomers display differences in their ability to activate the fungal pathogen Colletotrichum trifolii.

  2. Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors.

    PubMed

    Rönn, Robert; Gossas, Thomas; Sabnis, Yogesh A; Daoud, Hanna; Kerblom, Eva; Danielson, U Helena; Sandström, Anja

    2007-06-15

    There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P(1) C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.

  3. 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.

  4. 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.

  5. Xanosporic acid, an intermediate in bacterial degradation of the fungal phototoxin cercosporin.

    PubMed

    Mitchell, Thomas K; Alejos-Gonzalez, Fatima; Gracz, Hanna S; Danehower, David A; Daub, Margaret E; Chilton, William Scott

    2003-03-01

    The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers.

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

    PubMed

    Kaman, Wendy E; Voskamp-Visser, Ingrid; de Jongh, Denise M C; Endtz, Hubert P; van Belkum, Alex; Hays, John P; Bikker, Floris J

    2013-10-01

    Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.

  7. A sulfuric-lactic acid process for efficient purification of fungal chitosan with intact molecular weight.

    PubMed

    Naghdi, Mitra; Zamani, Akram; Karimi, Keikhosro

    2014-02-01

    The most recent method of fungal chitosan purification, i.e., two steps of dilute sulfuric acid treatment, pretreatment of cell wall at room temperature for phosphate removal and extraction of chitosan from the phosphate free cell wall at high temperature, significantly reduces the chitosan molecular weight. This study was aimed at improvement of this method. In the pretreatment step, to choose the best conditions, cell wall of Rhizopus oryzae, containing 9% phosphate, 10% glucosamine, and 21% N-acetyl glucosamine, was treated with sulfuric, lactic, acetic, nitric, or hydrochloric acid, at room temperature. Sulfuric acid showed the best performance in phosphate removal (90%) and cell wall recovery (89%). To avoid depolymerisation of chitosan, hot sulfuric acid extraction was replaced with lactic acid treatment at room temperature, and a pure fungal chitosan was obtained (0.12 g/g cell wall). Similar pretreatment and extraction processes were conducted on pure shrimp chitosan and resulted in a chitosan recovery of higher than 87% while the reduction of chitosan viscosity was less than 15%. Therefore, the sulfuric-lactic acid method purified the fungal chitosan without significant molecular weight manipulation.

  8. Distinguishing Functional Amino Acid Covariation from Background Linkage Disequilibrium in HIV Protease and Reverse Transcriptase

    PubMed Central

    Wang, Qi; Lee, Christopher

    2007-01-01

    Correlated amino acid mutation analysis has been widely used to infer functional interactions between different sites in a protein. However, this analysis can be confounded by important phylogenetic effects broadly classifiable as background linkage disequilibrium (BLD). We have systematically separated the covariation induced by selective interactions between amino acids from background LD, using synonymous (S) vs. amino acid (A) mutations. Covariation between two amino acid mutations, (A,A), can be affected by selective interactions between amino acids, whereas covariation within (A,S) pairs or (S,S) pairs cannot. Our analysis of the pol gene — including the protease and the reverse transcriptase genes — in HIV reveals that (A,A) covariation levels are enormously higher than for either (A,S) or (S,S), and thus cannot be attributed to phylogenetic effects. The magnitude of these effects suggests that a large portion of (A,A) covariation in the HIV pol gene results from selective interactions. Inspection of the most prominent (A,A) interactions in the HIV pol gene showed that they are known sites of independently identified drug resistance mutations, and physically cluster around the drug binding site. Moreover, the specific set of (A,A) interaction pairs was reproducible in different drug treatment studies, and vanished in untreated HIV samples. The (S,S) covariation curves measured a low but detectable level of background LD in HIV. PMID:17726544

  9. 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.

  10. Characterization of Five Fungal Endophytes Producing Cajaninstilbene Acid Isolated from Pigeon Pea [Cajanus cajan (L.) Millsp.

    PubMed Central

    Zu, Yuan Gang; Fu, Yu Jie; Wang, Wei; Luo, Meng; Efferth, Thomas

    2011-01-01

    Five fungal endophytes (K4, K5, K6, K9, K14) producing Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) were isolated from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]. CSA is responsible for the prominent pharmacological activities in pigeon pea. The amount of CSA in culture solution varied among the five fungal endophytes. K4 produced the highest levels of CSA (1037.13 µg/L) among the endophytes tested after incubation for five days. Both morphological characteristics and molecular methods were used for species identification of fungal endophytes. The five endophytic isolates were characterized by analyzing the internal transcribed spacer (ITS) rRNA and β-tubulin genes. The K4, K5, K9 and K14 strains isolated from pigeon pea roots were found to be closely related to the species Fusarium oxysporum. K6 was identified as Neonectria macrodidym. The present study is the first report on the isolation and identification of fungal endophytes producing CSA in pigeon pea. The study also provides a scientific base for large scale production of CSA. PMID:22102911

  11. 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

  12. 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.

  13. 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

  14. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

    PubMed Central

    Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

    1997-01-01

    The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

  15. Effect of exchange of amino acid residues of the surface region of the PST-01 protease on its organic solvent-stability.

    PubMed

    Ogino, Hiroyasu; Uchiho, Takeshi; Doukyu, Noriyuki; Yasuda, Masahiro; Ishimi, Kosaku; Ishikawa, Haruo

    2007-07-13

    The PST-01 protease from an organic solvent tolerant Pseudomonas aeruginosa has high stability and activity in the presence of various organic solvents. The structure gene of the PST-01 protease was amplified by the error-prone PCR method. The mutated proteases were incubated in the presence of acetonitrile. By measuring remaining activities, two kinds of mutated PST-01 proteases of which the stabilities were changed were selected. These mutations hardly changed the profile of the activity and stability at various pHs. Their activity and stability at higher temperatures were slightly lower than those of the wild-type PST-01 protease. The stabilities of the mutated enzymes in the presence of various organic solvents were greatly reduced. In both the mutated PST-01 proteases, amino acids located at the surface of the enzyme had been substituted.

  16. 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.

  17. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  18. Limestone dissolution induced by fungal mycelia, acidic materials, and carbonic anhydrase from fungi.

    PubMed

    Li, Wei; Zhou, Peng-Peng; Jia, Li-Ping; Yu, Long-Jiang; Li, Xue-Li; Zhu, Min

    2009-01-01

    Microorganisms influence the dissolution of a number of minerals. Limestone is one of the most abundant rock types in karst areas, and is predominantly calcium carbonate. Two types of experimental systems were designed in this paper, to make comparisons of limestone dissolution rate among the acidic materials and extracellular carbonic anhydrase (CA) excreted by fungi and the enwrapping effect of fungal mycelia. One was the simulated experimental system containing microorganisms. Another was the simulated experimental system without microorganisms. Results of previous experiment indicated that the acidic materials and CA like enzymatic materials excreted by fungi and the enwrapping effect of fungal mycelia were important factors influencing limestone dissolution. In the three factors mentioned above, the dissolution effect was mycelia enwraping effect>acidic dissolution effect>CA enzymatic effect. The results of the second experiment demonstrated further that the limestone dissolution effect of the acidic materials excreted by fungi was stronger than that of CA excreted by fungi. Nevertheless, CA still played an important role in promoting the dissolution of limestone.

  19. Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

    PubMed Central

    Ohno, Misa; Kimura, Masahiro; Miyazaki, Haruko; Okawa, Kazuaki; Onuki, Riho; Nemoto, Chiyuki; Tabata, Eri; Wakita, Satoshi; Kashimura, Akinori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Nukina, Nobuyuki; Bauer, Peter O.; Oyama, Fumitaka

    2016-01-01

    Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy. PMID:27883045

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

    PubMed

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

    2011-01-01

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

  1. 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

  2. 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.

  3. 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.

  4. Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia.

    PubMed

    Forlani, Giuseppe; Klimek-Ochab, Magdalena; Jaworski, Jakub; Lejczak, Barbara; Picco, Anna M

    2006-12-01

    Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.

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

    PubMed

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

    2014-03-11

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

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

    PubMed

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

    2017-02-01

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

  7. 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.

  8. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  9. 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.

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

    PubMed

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

    2016-03-01

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

  11. 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.

  12. 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%).

  13. Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

    PubMed Central

    Badgujar, Shamkant B.; Mahajan, Raghunath T.

    2013-01-01

    A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing. PMID:23476742

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

    PubMed

    Rout, Manoj Kumar; Hosur, Ramakrishna V

    2009-02-01

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

  15. Pinus pinaster seedlings and their fungal symbionts show high plasticity in phosphorus acquisition in acidic soils.

    PubMed

    Ali, M A; Louche, J; Legname, E; Duchemin, M; Plassard, C

    2009-12-01

    Young seedlings of maritime pine (Pinus pinaster Soland in Aït.) were grown in rhizoboxes using intact spodosol soil samples from the southwest of France, in Landes of Gascogne, presenting a large variation of phosphorus (P) availability. Soils were collected from a 93-year-old unfertilized stand and a 13-year-old P. pinaster stand with regular annual fertilization of either only P or P and nitrogen (N). After 6 months of culture in controlled conditions, different morphotypes of ectomycorrhiza (ECM) were used for the measurements of acid phosphatase activity and molecular identification of fungal species using amplification of the ITS region. Total biomass, N and P contents were measured in roots and shoots of plants. Bicarbonate- and NaOH-available inorganic P (Pi), organic P (Po) and ergosterol concentrations were measured in bulk and rhizosphere soil. The results showed that bulk soil from the 93-year-old forest stand presented the highest Po levels, but relatively higher bicarbonate-extractable Pi levels compared to 13-year-old unfertilized stand. Fertilizers significantly increased the concentrations of inorganic P fractions in bulk soil. Ergosterol contents in rhizosphere soil were increased by fertilizer application. The dominant fungal species was Rhizopogon luteolus forming 66.6% of analysed ECM tips. Acid phosphatase activity was highly variable and varied inversely with bicarbonate-extractable Pi levels in the rhizosphere soil. Total P or total N in plants was linearly correlated with total plant biomass, but the slope was steep only between total P and biomass in fertilized soil samples. In spite of high phosphatase activity in ECM tips, P availability remained a limiting nutrient in soil samples from unfertilized stands. Nevertheless young P. pinaster seedlings showed a high plasticity for biomass production at low P availability in soils.

  16. 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.

  17. 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.

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

    PubMed Central

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

    2016-01-01

    Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: 20.14±1.71 kg) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (p<0.05) the digestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (p<0.05) in the diet supplemented with CC protease. In Exp. 2, 12 crossbred barrows (initial body weight: 20.79±1.94 kg), fitted with T-cannulas at the distal ileum, were blocked by body weight into 2 groups with 6 pigs each. The diets were the same as those in Exp. 1. The CC protease increased (p<0.05) the AID of crude protein and some essential AA including arginine, isoleucine and leucine. The AID and ATTD of energy and nutrients had been improved (p<0.05) by supplemental CC protease, but the hindgut digestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment. PMID:27004811

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

    PubMed

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

    2016-12-01

    Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: 20.14±1.71 kg) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (p<0.05) the digestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (p<0.05) in the diet supplemented with CC protease. In Exp. 2, 12 crossbred barrows (initial body weight: 20.79±1.94 kg), fitted with T-cannulas at the distal ileum, were blocked by body weight into 2 groups with 6 pigs each. The diets were the same as those in Exp. 1. The CC protease increased (p<0.05) the AID of crude protein and some essential AA including arginine, isoleucine and leucine. The AID and ATTD of energy and nutrients had been improved (p<0.05) by supplemental CC protease, but the hindgut digestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment.

  20. 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.

  1. Engineering fungal de novo fatty acid synthesis for short chain fatty acid production

    PubMed Central

    Gajewski, Jan; Pavlovic, Renata; Fischer, Manuel; Boles, Eckhard; Grininger, Martin

    2017-01-01

    Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker's yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker's yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker's yeast, and can be plugged into essentially all currently available FA overproducing microorganisms. PMID:28281527

  2. 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.

  3. 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.

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

    PubMed Central

    Wagner, M K; Busta, F F

    1985-01-01

    The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP. PMID:2992374

  5. Dry fermented sausages made with a protease from Aspergillus oryzae and/or a starter culture.

    PubMed

    Zapelena, M J; Astiasarán, I; Bello, J

    1999-08-01

    Sausages with a starter culture (Lactobacillus plantarum + Staphylococcus carnosus), a protease (Fungal Protease from Solvay Enzymes) and both enzyme/starter were produced from the same raw matter under the same conditions for 15 days of ripening. The lowest pH values were found in Fungal/starter sausages. Significantly higher amounts of free amino acids (FAA) were seen in sausages containing enzyme from the 3rd day of ripening, whereas amino acids from peptides (PAA) were significantly higher only after 15 days of ripening. Enzyme addition gave rise to changes in 10 of the 15 FAA analyzed. Histidine was the main amino acid from the peptide fraction that increased in both sausages containing added enzyme. Although trained panelists detected some sensorial benefits in the sausages with added enzyme, the effects were not as marked as might have been expected.

  6. 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.

  7. 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

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

    PubMed

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

    2014-05-01

    Although type 2 immune responses to environmental Ags 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. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. 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 naive 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.

  9. An additional Meyerozyma guilliermondii IMH3 gene confers mycophenolic acid resistance in fungal CTG clade species.

    PubMed

    Defosse, Tatiana A; Mélin, Céline; Clastre, Marc; Besseau, Sébastien; Lanoue, Arnaud; Glévarec, Gaëlle; Oudin, Audrey; Dugé de Bernonville, Thomas; Vandeputte, Patrick; Linder, Tomas; Bouchara, Jean-Philippe; Courdavault, Vincent; Giglioli-Guivarc'h, Nathalie; Papon, Nicolas

    2016-09-01

    The fungal CTG clade comprises a number of well-known yeasts that impact human health or with high biotechnological potential. To further extend the set of molecular tools dedicated to these microorganisms, the initial focus of this study was to develop a mycophenolic acid (MPA) resistance cassette. Surprisingly, while we were carrying out preliminary susceptibility testing experiments in a set of yeast species, Meyerozyma guilliermondii, although not being a MPA producer, was found to be primarily resistant toward this drug, whereas a series of nine related species were susceptible to MPA. Using comparative and functional genomic approaches, we demonstrated that all MPA-susceptible CTG clade species display a single gene, referred to as IMH3.1, encoding the MPA target inosine monophosphate dehydrogenase (IMPDH) and that MPA resistance relies on the presence in the M. guilliermondii genome of an additional IMPDH-encoding gene (IMH3.2). The M. guilliermondii IMH3.2 gene displays marked differences compared to IMH3.1 including the lack of intron, a roughly 160-fold higher transcription level and a serine residue at position 251. Placed under the control of the M. guilliermondii actin 1 gene promoter, IMH3.2 was successfully used to transform Lodderomyces elongisporus, Clavispora lusitaniae, Scheffersomyces stipitis and Candida parapsilosis.

  10. 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.

  11. Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus.

    PubMed Central

    Stennicke, H. R.; Birktoft, J. J.; Breddam, K.

    1996-01-01

    The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1. PMID:8931145

  12. 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.

  13. 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.

  14. Conditions influencing the synthesis of acid protease by Mucor pusillus Lindt.

    PubMed

    Somkuti, G A; Babel, F J

    1967-11-01

    Protease synthesis by Mucor pusillus Lindt, in a wheat bran medium under submerged conditions, was influenced by substrate concentration, initial pH of the medium, and temperature of incubation. A 4% wheat bran (dry weight) concentration was satisfactory for enzyme production. The initial pH of the medium had a substantial effect on enzyme synthesis; adjustment of the enzyme production medium to pH 5.0 prior to sterilization was desirable. Incubation at 35 C resulted in the best enzyme yields. Under optimal conditions of enzyme production, maximal activity was detected after 5 days of incubation. The enrichment of the medium with glucose increased the yield of mycelia but lowered the amount of enzyme produced.

  15. 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.

  16. 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.

  17. Generation of the volatile spiroketals conophthorin and chalcogran by fungal spores on polyunsaturated fatty acids common to almonds and pistachios.

    PubMed

    Beck, John J; Mahoney, Noreen E; Cook, Daniel; Gee, Wai S

    2012-12-05

    The spiroketal (E)-conophthorin has recently been reported as a semiochemical of the navel orangeworm moth, a major insect pest of California pistachios and almonds. Conophthorin and the isomeric spiroketal chalcogran are most commonly known as semiochemicals of several scolytid beetles. Conophthorin is both an insect- and plant-produced semiochemical widely recognized as a nonhost plant volatile from the bark of several angiosperm species. Chalcogran is the principal aggregation pheromone component of the six-spined spruce bark beetle. Recent research has shown conophthorin is produced by almonds undergoing hull-split, and both spiroketals are produced by mechanically damaged almonds. To better understand the origin of these spiroketals, the volatile emissions of orchard fungal spores on fatty acids common to both pistachios and almonds were evaluated. The volatile emission for the first 13 days of spores placed on a fatty acid was monitored. The spores investigated were Aspergillus flavus (atoxigenic), A. flavus (toxigenic), Aspergillus niger, Aspergillus parasiticus, Penicillium glabrum, and Rhizopus stolonifer. The fatty acids used as growth media were palmitic, oleic, linoleic, and linolenic. Spores on linoleic acid produced both spiroketals, those on linolenic acid produced only chalcogran, and those on palmitic and oleic acid did not produce either spiroketal. This is the first report of the spiroketals conophthorin and chalcogran from a fungal source.

  18. 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.

  19. 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

  20. 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.

  1. Transfecting deoxyribonucleic acid of Bacillus bacteriophage phi 29 that is protease sensitive.

    PubMed

    Hirokawa, H

    1972-06-01

    The transfecting activity of Bacillus phage varphi29 DNA, extracted either by sodium lauroyl sarcosine-phenol or by 2 M perchlorate, was destroyed by treatment with proteolytic enzymes, although these enzymes did not effect transfecting DNAs of SPP1, SPO1, and SP50. These facts suggest that a protein is associated with transfective varphi29 DNA. Stabilization of protease-resistance during transfection appeared earlier than that of DNaseresistance, indicating that the protein associated with varphi29 DNA is necessary for initiation of the incorporation of DNA molecules into competent cells. The physical nature of varphi29 DNA before and after the trypsin treatment was investigated by sucrose and CsCl density gradient centrifugations. The trypsin treatment did not alter the sedimentation rate of the unit varphi29 DNA; however, it did convert the sedimentation rate of the aggregated material in the untreated DNA to that of the unit varphi29 DNA. The density of the trypsinized DNA was 0.009 g/cm(3) greater than that of the untreated DNA. The possible location of the protein on the DNA is discussed.

  2. 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.

  3. 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.

  4. 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.

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

    PubMed

    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.

  6. 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.

  7. HIV-Protease Inhibitors Suppress Skeletal Muscle Fatty Acid Oxidation by Reducing CD36 and CPT-I Fatty Acid Transporters

    PubMed Central

    Richmond, Scott R.; Carper, Michael J.; Lei, Xiaoyong; Zhang, Sheng; Yarasheski, Kevin E.; Ramanadham, Sasanka

    2010-01-01

    Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5 μmol/l) plus 20 μmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase I (CPTI) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV. PMID:20117238

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

    PubMed Central

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

    1989-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Hidayat, Habibi

    2017-03-01

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

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

    PubMed Central

    Patel, Mitesh; Mandava, Nanda; Gokulgandhi, Mitan; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2. PMID:24727459

  11. Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events.

    PubMed

    McCarthy, A D; Goldring, J P; Hardie, D G

    1983-10-17

    The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium [3H]borohydride. Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein. The results show that the enoyl reductase lies between the 3-oxoacylsynthase and the acyl carrier. This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events.

  12. Effect of solvent on the conformation and dynamics of Aspartic Acid Protease by a coarse-grained bond-fluctuating Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Pandey, Ras; Farmer, Barry

    2007-03-01

    In a coarse-grained description of a protein chain, all of the 20 amino acid residues can be broadly divided into three groups, hydrophobic (H), polar (P), and electrostatic (E). A protein can be described by tethered nodes in a chain with a node representing the amino acid group. Aspartic acid protease consists of 99 residues in a well-defined sequence. The specific sequence of H, P and E nodes tethered together by fluctuating bonds is placed on a cubic lattice where empty lattice sites constitute an effective solvent medium. The amino groups (nodes) interact with the solvent (S) sites with appropriate attractive (HS) and repulsive (PS) interactions with the solvent. Each node executes its stochastic movement with the Metropolis algorithm. Variations of the root mean square displacements of the center of mass and that of its center node of the protease chain, and its gyration radius with the time steps are examined for different solvent strength. The structure of the protease swells on increasing the solvent interaction strength which tends to enhance the relaxation time to reach diffusive behavior of the chain.

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

    NASA Astrophysics Data System (ADS)

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

    2008-03-01

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

  14. 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)

  15. 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

  16. 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.

  17. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T).

    PubMed

    Kurata, Atsushi; Uchimura, Kohsuke; Shimamura, Shigeru; Kobayashi, Tohru; Horikoshi, Koki

    2007-11-01

    The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T), was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0-9.5 and 45 degrees C in 100 mM glycine-NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40(T) was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

  18. Protease Profiling in Prostate Cancer

    DTIC Science & Technology

    2004-05-01

    acid synthase, which contains a serine hydrolase domain. We identified a lead inhibitor of this domain of fatty acid synthase, called Orlistat, which...SUBJECT TERMS 15. NUMBER OF PAGES Prostate cancer, tumor biology, protease, proteomics, transgenic, 20 animal model, fatty acid synthase, orlistat 16...the enzymes we identified is fatty acid synthase. Fatty acid synthase is the sole enzyme responsible for the cellular synthesis of fatty acids . This

  19. 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...

  20. 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.

  1. Enzymatic hydrolysis of cuttlefish (Sepia officinalis) and sardine (Sardina pilchardus) viscera using commercial proteases: effects on lipid distribution and amino acid composition.

    PubMed

    Kechaou, Emna Soufi; Dumay, Justine; Donnay-Moreno, Claire; Jaouen, Pascal; Gouygou, Jean-Paul; Bergé, Jean-Pascal; Amar, Raja Ben

    2009-02-01

    Total lipid and phospholipid recovery as well as amino acid quality and composition from cuttlefish (Sepia officinalis) and sardine (Sardina pilchardus) were compared. Enzymatic hydrolyses were performed using the three proteases Protamex, Alcalase, and Flavourzyme by the pH-stat method (24 h, pH 8, 50 degrees C). Three fractions were generated: an insoluble sludge, a soluble aqueous phase, and an oily phase. For each fraction, lipids, phospholipids, and proteins were quantified. Quantitative and qualitative analyses of the raw material and hydrolysates were performed. The degree of hydrolysis (DH) for cuttlefish viscera was 3.2% using Protamex, 6.8% using Flavourzyme, and 7% using Alcalase. DH for sardine viscera was 1.9% (using Flavourzyme), 3.1% (using Protamex) and 3.3% (using Alcalase). Dry matter yields of all hydrolysis reactions increased in the aqueous phases. Protein recovery following hydrolysis ranged from 57.2% to 64.3% for cuttlefish and 57.4% to 61.2% for sardine. Tissue disruption following protease treatment increased lipid extractability, leading to higher total lipid content after hydrolysis. At least 80% of the lipids quantified in the raw material were distributed in the liquid phases for both substrates. The hydrolysed lipids were richer in phospholipids than in the lipids extracted by classical chemical extraction, especially after Flavourzyme hydrolysis for cuttlefish and Alcalase hydrolysis for sardine. The total amino acid content differed according to the substrate and the enzyme used. However, regardless of the raw material or the protease used, hydrolysis increased the level of essential amino acids in the hydrolysates, thereby increasing their potential nutritional value for feed products.

  2. 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.

  3. 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.

  4. 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.

  5. Gallic acid and tannase accumulation during fungal solid state culture of a tannin-rich desert plant (Larrea tridentata Cov.).

    PubMed

    Treviño-Cueto, B; Luis, M; Contreras-Esquivel, J C; Rodríguez, R; Aguilera, A; Aguilar, C N

    2007-02-01

    Larrea tridentata (Sesse & Mocino ex DC.) Coville, also known as Larrea, gobernadora, chaparral, or creosote bush, is a shrubby plant which dominates some areas of the desert southwest in the United States and Northern Mexico and its use has not been exploited and standardized. In this study, gobernadora was studied to evaluate its potential use for support of solid state culture. Influence of two minimal media added with gobernadora powder as the sole carbon source and inducer of tannin-degrading enzymes was evaluated. Cultures were initially 70% moisture, had a pH of 5.5 and were inoculated with Aspergillus niger Aa-20 at 2 x 10(7) spores per gram of media. Analysis of pH, moisture, tannin uptake, gallic acid accumulation and tannase production were evaluated. Results indicated a high content of condensed (39.4%dm) and hydrolysable (22.8%dm) tannins. Invasion capacity of fungal growth was of 0.15 mmh(-1). Tannase production reached values of 1040 Ul(-1) at 43 h of culture. During the first 48 h of culture, the concentration of gallic acid accumulation was 0.33 gl(-1). Gobernadora is a potential source of gallic acid and tannase production by solid state culture; however, further optimization of the process is needed.

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

    NASA Astrophysics Data System (ADS)

    Pandey, Ras; Farmer, Barry

    2008-03-01

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

  7. 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

  8. Proteases from psychrotrophs: an overview.

    PubMed

    Kasana, Ramesh Chand

    2010-05-01

    Proteases are hydrolytic enzymes which catalyze the total hydrolysis of proteins in to amino acids. Although proteolytic enzymes can be obtained from animals and plants but microorganisms are the preferred source for industrial applications in view of scientific and economical advantage. Among various groups of microbes, psychrotrophs are ideal candidates for enzymes production keeping in mind that enzymes active at low temperature and stable under alkaline condition, in presence of oxidants and detergents are in large demand as laundry additive. The proteases from psychrotrophs also find application in environmental bioremediation, food and molecular biology. During the previous two decades, proteases from psychrotrophs have received increased attention because of their wide range of applications, but the full potential of psychrotrophic proteases has not been exploited. This review focuses attention on the present status of knowledge on the production, optimization, molecular characteristics, applications, substrate specificity, and crystal structure of psychrotrophic proteases. The review will help in making strategies for exploitation of psychrotrophic protease resources and improvement of enzymes to obtain more robust proteases of industrial and biotechnological significance.

  9. The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases.

    PubMed

    Shokhen, Michael; Khazanov, Netaly; Albeck, Amnon

    2007-08-13

    What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pKa of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site--the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.

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

    PubMed

    Najim, N; Aryana, Kayanush J

    2013-06-01

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

  11. 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.

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

    PubMed

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

    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.

  13. 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

  14. 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

  15. Acid phosphatase positional correlations in solid surface fungal cultivation: a fractal interpretation of biochemical differentiation.

    PubMed

    Jones, C L; Lonergan, G T; Mainwaring, D E

    1995-03-28

    Colour image analysis was used to measure the positional correlation between acid phosphatase intracellular concentration and hyphal cellular differentiation which leads to concentric circular zonal activity patterns. Acid phosphatase is strongly implicated in the biochemical control of hyphal branching, and exo-enzyme secretion, such as laccase in fungi, occurs predominately via the hyphal tips. Different concentrations of an organic dye were used to effect substrate induction of the enzyme response, which was shown to be statistically correlated according to a fractal power law (H approximately 0.39). A self-organized critical state for the molecular responsivity of dissipative enzyme expression is hypothesized as an efficient mechanism for hyphal adaptation, also accounting for the underlying biophysics of the observed pattern formations.

  16. 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.

  17. 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

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

    PubMed Central

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

    2013-01-01

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

  19. Salicylic acid stimulates secretion of the normally symplastic enzyme mannitol dehydrogenase: a possible defense against mannitol-secreting fungal pathogens.

    PubMed

    Cheng, Fang-yi; Zamski, Eli; Guo, Wei-wen; Pharr, D Mason; Williamson, John D

    2009-11-01

    The sugar alcohol mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, mannitol is reported to be an antioxidant and as such may play a role in host-pathogen interactions. Current research suggests that pathogenic fungi can secrete mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, mannitol dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against mannitol-secreting fungal pathogens such as Alternaria. After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant's response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.

  20. 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

  1. 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.

  2. 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.

  3. Biofluid proteases profiling in diabetes mellitus.

    PubMed

    Trindade, Fábio; Ferreira, Rita; Amado, Francisco; Vitorino, Rui

    2015-01-01

    The investigation of protease relevance in biologic systems beyond catabolism of proteins and peptides to amino acids has stimulated interest as to their role in the pathogenesis of several disorders including diabetes mellitus (DM). Evaluation of proteases and the assessment of their activity in biofluids are fundamental to elucidate these proteolytic systems in DM and its related complications. In contrast to traditional immunoassay or substrate based approaches that targeted specific proteases and their inhibitors, the field of degradomics has provided a comprehensive approach to study these enzymes. Although the degradome contains over 500 proteases, very few have been associated with DM and its micro- and macrovascular complications. In this paper, we review these proteases and their respective inhibitors with emphasis on DM. It is likely that future research will expand these initial studies and look to develop high throughput automated technologies to identify and characterize biofluid proteases of diagnostic and prognostic value in other pathologies.

  4. 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.

  5. Fungal melanonychia.

    PubMed

    Finch, Justin; Arenas, Roberto; Baran, Robert

    2012-05-01

    Fungal melanonychia is a relatively rare nail disorder caused by nail infection that produces brown-to-black pigmentation of the nail unit. The number of organisms implicated as etiologic agents of fungal melanonychia is increasing, and the list currently tops 21 species of dematiaceous fungi and at least 8 species of nondematiaceous fungi. These superficial infections may clinically mimic subungual melanoma and are often not responsive to traditional antifungal therapy. This article reviews the literature on fungal melanonychia and the role of fungal melanin in infection.

  6. L-chicoric acid, an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350).

    PubMed

    Robinson, W E

    1998-08-01

    Combinations of anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have proven immensely potent in the therapy of acquired immune deficiency syndrome (AIDS). To determine whether HIV integrase is a suitable target for combination therapy, the ability of an HIV integrase inhibitor, L-chicoric acid, to work in combination with a protease inhibitor and Zidovudine was tested in vitro. The addition of L-chicoric acid to either Zidovudine or protease inhibitor improved upon the observed anti-HIV activity of either compound alone. When all three drugs were combined, the anti-HIV activity was substantially better than either of the three compounds alone or any combination of two inhibitors. Doses of both Zidovudine and protease inhibitor could be reduced by more than 33% for an equivalent anti-HIV effect if L-chicoric acid was added. The improved anti-HIV activity was observed with a tissue culture adapted strain of HIV (HIV(LAI)) and with limited passage clinical isolates of HIV (HIV(R19) and HIV(R45)). These data demonstrate that a first generation HIV integrase inhibitor, L-chicoric acid, is at least additive in combination with existing multi-drug regimens and suggest that HIV integrase will be an excellent target for combination therapy of HIV infection.

  7. Metabolomics analysis of fungal biofilm development and of arachidonic acid-based quorum sensing mechanism.

    PubMed

    Ząbek, Adam; Junka, Adam; Szymczyk, Patrycja; Wojtowicz, Wojciech; Klimek-Ochab, Magdalena; Młynarz, Piotr

    2017-04-03

    The infections caused by filamentous fungi are becoming worldwide problem of healthcare systems due to increasing drug-resistance of this microorganism and increasing number of immunocompromised nosocomial patients. These infections are related with Aspergillus ability to form sessile communities referred to as the biofilms. The small compounds known as quorum sensing (QS) molecules allow this microorganism to coordinate all processes taking place during biofilm formation and maturation. In the study presented, the HRMAS (1) H NMR metabolomic approach was applied to define composition of extra and intracellular metabolites produced by biofilmic and planktonic (aka free-swimming) cultures of this microorganism and to evaluate impact of quorum sensing molecule, arachidonic acid (AA) on biofilm formation. The Scanning Electron Microscopy was used to confirm Aspergillus ability to form biofilm in vitro, while multivariate and univariate data analysis was applied to analyze data obtained. The Aspergillus strain was able to form strong biofilm structures in vitro. The statistical analysis revealed significant changes of metabolite production depending on Aspergillus culture type (biofilm vs. plankton), time and presence of QS molecules. The data obtained, if developed, might be used in future NMR diagnostics as markers of Aspergillus biofilm-related infections and lead to shorten time between pathogen identification and introduction of treatment.

  8. Endohyphal Bacterium Enhances Production of Indole-3-Acetic Acid by a Foliar Fungal Endophyte

    PubMed Central

    Hoffman, Michele T.; Gunatilaka, Malkanthi K.; Wijeratne, Kithsiri; Gunatilaka, Leslie; Arnold, A. Elizabeth

    2013-01-01

    Numerous plant pathogens, rhizosphere symbionts, and endophytic bacteria and yeasts produce the important phytohormone indole-3-acetic acid (IAA), often with profound effects on host plants. However, to date IAA production has not been documented among foliar endophytes -- the diverse guild of primarily filamentous Ascomycota that live within healthy, above-ground tissues of all plant species studied thus far. Recently bacteria that live within hyphae of endophytes (endohyphal bacteria) have been detected, but their effects have not been studied previously. Here we show not only that IAA is produced in vitro by a foliar endophyte (here identified as Pestalotiopsis aff. neglecta, Xylariales), but that IAA production is enhanced significantly when the endophyte hosts an endohyphal bacterium (here identified as Luteibacter sp., Xanthomonadales). Both the endophyte and the endophyte/bacterium complex appear to rely on an L-tryptophan dependent pathway for IAA synthesis. The bacterium can be isolated from the fungus when the symbiotic complex is cultivated at 36°C. In pure culture the bacterium does not produce IAA. Culture filtrate from the endophyte-bacterium complex significantly enhances growth of tomato in vitro relative to controls and to filtrate from the endophyte alone. Together these results speak to a facultative symbiosis between an endophyte and endohyphal bacterium that strongly influences IAA production, providing a new framework in which to explore endophyte-plant interactions. PMID:24086270

  9. Enhanced Production of Itaconic Acid through Development of Transformed Fungal Strains of Aspergillus terreus.

    PubMed

    Shin, Woo-Shik; Park, Boonyoung; Lee, Dohoon; Oh, Min-Kyu; Chun, Gie-Taek; Kim, Sangyong

    2017-02-28

    Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.

  10. A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity.

    PubMed

    Teder, Tarvi; Boeglin, William E; Schneider, Claus; Brash, Alan R

    2017-03-28

    The genome of the fungal plant pathogen Fusarium graminearum harbors six catalases, one of which has the sequence characteristics of a fatty acid peroxide-metabolizing catalase. We cloned and expressed this hemoprotein (designated as Fg-cat) along with its immediate neighbor, a 13S-lipoxygenase (cf. Brodhun et al., PloS One, e64919, 2013) that we considered might supply a fatty acid hydroperoxide substrate. Indeed, Fg-cat reacts abruptly with the 13S-hydroperoxide of linoleic acid (13S-HPODE) with an initial rate of 700-1300s(-1). By comparison there was no reaction with 9R- or 9S-HPODEs and extremely weak reaction with 13R-HPODE (~0.5% of the rate with 13S-HPODE). Although we considered Fg-cat as a candidate for the allene oxide synthase of the jasmonate pathway in fungi, the main product formed from 13S-HPODE was identified by UV, MS, and NMR as 9-oxo-10E-12,13-cis-epoxy-octadecenoic acid (with no traces of AOS activity). The corresponding analog is formed from the 13S-hydroperoxide of α-linolenic acid along with novel diepoxy-ketones and two C13 aldehyde derivatives, the reaction mechanisms of which are proposed. In a peroxidase assay monitoring the oxidation of ABTS, Fg-cat exhibited robust activity (kcat 550s(-1)) using the 13S-hydroperoxy-C18 fatty acids as the oxidizing co-substrate. There was no detectable peroxidase activity using the corresponding 9S-hydroperoxides, nor with t-butyl hydroperoxide, and very weak activity with H2O2 or cumene hydroperoxide at micromolar concentrations of Fg-cat. Fg-cat and the associated lipoxygenase gene are present together in fungal genera Fusarium, Metarhizium and Fonsecaea and appear to constitute a partnership for oxidations in fungal metabolism or defense.

  11. Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

    PubMed Central

    2014-01-01

    Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

  12. 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.

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    2015-10-01

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

  15. Amino Acid Starvation Induced by Protease Inhibition Produces Differential Alterations in Redox Status and the Thiol Proteome in Organogenesis-Stage Rat Embryos and Visceral Yolk Sacs

    PubMed Central

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

    2015-01-01

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

  16. Fungal hemolysins

    PubMed Central

    Nayak, Ajay P.; Green, Brett J.; Beezhold, Donald H.

    2015-01-01

    Hemolysins are a class of proteins defined by their ability to lyse red cells but have been described to exhibit pleiotropic functions. These proteins have been extensively studied in bacteria and more recently in fungi. Within the last decade, a number of studies have characterized fungal hemolysins and revealed a fascinating yet diverse group of proteins. The purpose of this review is to provide a synopsis of the known fungal hemolysins with an emphasis on those belonging to the aegerolysin protein family. New insight and perspective into fungal hemolysins in biotechnology and health are additionally presented. PMID:22769586

  17. 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.

  18. Cytomegalovirus protease targeted prodrug development.

    PubMed

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

    2013-04-01

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

  19. Fungal Tests

    MedlinePlus

    ... diagnosis is needed, as in cases of persistent, deep, or systemic infections, more extensive testing may be ... mouth (thrush) Vaginal itching and discharge (yeast infection) Deep and systemic fungal infections may cause a variety ...

  20. 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

  1. 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...

  2. 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)

  3. Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14*

    PubMed Central

    Lahaie, Nicolas; Kralikova, Michaela; Prézeau, Laurent; Blahos, Jaroslav; Bouvier, Michel

    2016-01-01

    Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b) subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABAB in a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination. PMID:26817839

  4. Rhb1 regulates the expression of secreted aspartic protease 2 through the TOR signaling pathway in Candida albicans.

    PubMed

    Chen, Yu-Ting; Lin, Chia-Ying; Tsai, Pei-Wen; Yang, Cheng-Yao; Hsieh, Wen-Ping; Lan, Chung-Yu

    2012-02-01

    Candida albicans is a major fungal pathogen in humans. In C. albicans, secreted aspartyl protease 2 (Sap2) is the most highly expressed secreted aspartic protease in vitro and is a virulence factor. Recent research links the small GTPase Rhb1 to C. albicans target of rapamycin (TOR) signaling in response to nitrogen availability. The results of this study show that Rhb1 is related to cell growth through the control of SAP2 expression when protein is the major nitrogen source. This process involves various components of the TOR signaling pathway, including Tor1 kinase and its downstream effectors. TOR signaling not only controls SAP2 transcription but also affects Sap2 protein levels, possibly through general amino acid control. DNA microarray analysis identifies other target genes downstream of Rhb1 in addition to SAP2. These findings provide new insight into nutrients, Rhb1-TOR signaling, and expression of C. albicans virulence factor.

  5. 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).

  6. Fungal polysaccharides.

    PubMed

    San-Blas, G; Suzuki, S; Hearn, V; Pinel, C; Kobayashi, H; Mendez, C; Niño, G; Nishikawa, A; San-Blas, F; Shibata, N

    1994-01-01

    Fungal polysaccharides are cell wall components which may act as antigens or as structural substrates. As antigens, the role of mannans in Saccharomyces cerevisiae and Candida albicans, and of glycoproteins in Aspergillus fumigatus are discussed. Analyses on beta-glucan synthetase in Paracoccidioides brasiliensis and the inhibitory effect of Hansenula mrakii killer toxin on beta-glucan biosynthesis are also considered.

  7. Fungal arthritis

    MedlinePlus

    ... A.D.A.M. Editorial team. Related MedlinePlus Health Topics Fungal Infections Infectious Arthritis Browse the Encyclopedia A.D.A.M., Inc. is accredited by URAC, also known as the American Accreditation HealthCare ... for online health information and services. Learn more about A.D. ...

  8. 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 ...

  9. Fungal Sinusitis

    MedlinePlus

    ... presence of large granules that attract the reddish-orange eosin stain) to attack fungi, and the eosinophils irritate the membranes in the nose. As long as fungi remain, so will the irritation. Chronic Indolent Sinusitis is an invasive form of fungal sinusitis in ...

  10. Fungal Entomopathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal entomopathogens are important biological control agents worldwide and have been the subject of intense research for more than100 years. They exhibit both sexual and asexual reproduction and produce different types of infective propagules. Their mode of action against insects involves attachme...

  11. Cyclopaldic acid, seiridin, and sphaeropsidin A as fungal phytotoxins, and larvicidal and biting deterrents against Aedes aegypti (Diptera: Culicidae): structure-activity relationships.

    PubMed

    Cimmino, Alessio; Andolfi, Anna; Avolio, Fabiana; Ali, Abbas; Tabanca, Nurhayat; Khan, Ikhlas A; Evidente, Antonio

    2013-07-01

    Aedes aegypti L. is the major vector of the arboviruses responsible for dengue fever, one of the most devastating human diseases. From a preliminary screening of fungal phytotoxins, cyclopaldic acid (1), seiridin (2), sphaeropsidin A (4), and papyracillic acid (5) were evaluated for their biting deterrent and larvicidal activities against Ae. aegypti L. Because compounds 1, 2, 4, and 5 exhibited mosquito biting deterrent activities and 1 and 4 demonstrated larvicidal activities, further structureactivity relationship studies were initiated on these toxins. In biting-deterrence bioassays, 1, 2, 4, and 5, 3,8-didansylhydrazone of cyclopaldic acid, 1F, 5-azidopentanoate of cyclopaldic acid A, 1G, the reduced derivative of cyclopaldic acid, 1 H, isoseiridin (3), 2'-O-acetylseiridin (2A), 2'-oxoseiridin (2C), 6-O-acetylsphaeropsidin A (4A), 8,14-methylensphaeropsidin A methyl ester (4B), and sphaeropsidin B (4C) showed activities higher than the solvent control. Sphaeropsidin B (4C) was the most active compound followed by 2A, while the other compounds were less active. Biting-deterrence activity of compound 4C was statistically similar to DEET. In the larvicidal screening bioassays, only compounds 1 and 4 demonstrated larvicidal activities. Based on LD50 values, compound 4 (LD50 36.8 ppm) was significantly more active than compound 1 (LD50 58.2 ppm). However, the activity of these compounds was significantly lower than permethrin.

  12. 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

  13. Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection.

    PubMed Central

    Ashbaugh, C D; Warren, H B; Carey, V J; Wessels, M R

    1998-01-01

    Human invasive soft-tissue infections caused by group A Streptococcus are associated with significant morbidity and mortality. To investigate the pathogenesis of these serious infections, we characterized the host response to bacterial challenge with an M-type 3 isolate recovered from a patient with necrotizing fasciitis, or with isogenic gene replacement mutants deficient in cysteine protease, hyaluronic acid capsule, or M protein in a murine model of human invasive soft-tissue infection. Animals challenged with the wild-type or cysteine protease-deficient strain developed spreading tissue necrosis at the site of inoculation, became bacteremic, and subsequently died. Histopathologic examination of the necrotic lesion revealed bacteria throughout inflamed subcutaneous tissue. Arterioles and venules in the subcutaneous layer were thrombosed and the overlying tissue was infarcted. In contrast, animals challenged with either an acapsular or M protein-deficient mutant developed a focal area of tissue swelling at the site of inoculation without necrosis or subsequent systemic disease. Histopathologic examination of the soft-tissue lesion demonstrated bacteria confined within a well-formed subcutaneous abscess. We conclude that the group A streptococcal hyaluronic acid capsule and M protein, but not the cysteine protease, are critical for the development of tissue necrosis, secondary bacteremia, and lethal infection in a murine model of human necrotizing fasciitis. PMID:9691092

  14. 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

  15. 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

  16. 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.

  17. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

  18. 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

  19. 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

  20. Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops.

    PubMed

    Wu, Jiang; Bian, Yan; Tang, Bing; Chen, Xiangdong; Shen, Ping; Peng, Zhenrong

    2004-01-30

    Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.

  1. Fungal nail infection

    MedlinePlus

    ... Common fungal infections include: Athlete's foot Jock itch Ringworm on the skin of the body or head ... fungal infection. Alternative Names Nails - fungal infection; Onychomycosis; Tinea unguium Images Nail infection, candidal Yeast and mold ...

  2. 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

  3. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway.

    PubMed

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD(+) requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP(+)/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s(-1), and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD(+)/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s(-1), and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism.

  4. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

    PubMed Central

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

  5. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Helvolic acid, an antibacterial nortriterpenoid from a fungal endophyte, Xylaria sp. of orchid Anoectochilus setaceus endemic to Sri Lanka

    PubMed Central

    Ratnaweera, Pamoda B.; Williams, David E.; de Silva, E. Dilip; Wijesundera, Ravi L.C.; Dalisay, Doralyn S.; Andersen, Raymond J.

    2014-01-01

    An endophytic fungus was isolated from surface sterilized leaf segments of Anoectochilus setaceus, an orchid endemic to Sri Lanka, and was identified as Xylaria sp. by morphological characters and DNA sequencing. Bioassay-guided chromatographic fractionation of the organic extract of a laboratory culture of this fungus led to the isolation of the known antibacterial helvolic acid. Helvolic acid was active against the Gram-positive bacteria, Bacillus subtilis [minimal inhibitory concentrations (MIC), 2 μg mL−1] and methicillin-resistant Staphylococcus aureus (MIC, 4 μg mL−1). PMID:24772371

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. [Chloroplast Deg proteases].

    PubMed

    Grabsztunowicz, Magda; Luciński, Robert; Baranek, Małgorzata; Sikora, Bogna; Jackowski, Grzegorz

    2011-01-01

    For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.

  18. Functional Divergence of Two Secreted Immune Proteases of Tomato.

    PubMed

    Ilyas, Muhammad; Hörger, Anja C; Bozkurt, Tolga O; van den Burg, Harrold A; Kaschani, Farnusch; Kaiser, Markus; Belhaj, Khaoula; Smoker, Matthew; Joosten, Matthieu H A J; Kamoun, Sophien; van der Hoorn, Renier A L

    2015-08-31

    Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

  19. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.

  20. 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

  1. The site-2 protease.

    PubMed

    Rawson, Robert B

    2013-12-01

    The site-2 protease (S2P) is an unusually-hydrophobic integral membrane protease. It cleaves its substrates, which are membrane-bound transcription factors, within membrane-spanning helices. Although structural information for S2P from animals is lacking, the available data suggest that cleavage may occur at or within the lipid bilayer. In mammalian cells, S2P is essential owing to its activation of the sterol regulatory element binding proteins (SREBPs); in the absence of exogenous lipid, cells lacking S2P cannot survive. S2P is also important in the endoplasmic reticulum (ER) stress response, activating several different membrane-bound transcription factors. Human patients harboring reduction-of-function mutations in S2P exhibit an array of pathologies ranging from skin defects to neurological abnormalities. Surprisingly, Drosophila melanogaster lacking S2P are viable and fertile. This article is part of a Special Issue entitled: Intramembrane Proteases.

  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. 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

  4. Impact of the microbial derived short chain fatty acid propionate on host susceptibility to bacterial and fungal infections in vivo

    PubMed Central

    Ciarlo, Eleonora; Heinonen, Tytti; Herderschee, Jacobus; Fenwick, Craig; Mombelli, Matteo; Le Roy, Didier; Roger, Thierry

    2016-01-01

    Short chain fatty acids (SCFAs) produced by intestinal microbes mediate anti-inflammatory effects, but whether they impact on antimicrobial host defenses remains largely unknown. This is of particular concern in light of the attractiveness of developing SCFA-mediated therapies and considering that SCFAs work as inhibitors of histone deacetylases which are known to interfere with host defenses. Here we show that propionate, one of the main SCFAs, dampens the response of innate immune cells to microbial stimulation, inhibiting cytokine and NO production by mouse or human monocytes/macrophages, splenocytes, whole blood and, less efficiently, dendritic cells. In proof of concept studies, propionate neither improved nor worsened morbidity and mortality parameters in models of endotoxemia and infections induced by gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae), gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae) and Candida albicans. Moreover, propionate did not impair the efficacy of passive immunization and natural immunization. Therefore, propionate has no significant impact on host susceptibility to infections and the establishment of protective anti-bacterial responses. These data support the safety of propionate-based therapies, either via direct supplementation or via the diet/microbiota, to treat non-infectious inflammation-related disorders, without increasing the risk of infection. PMID:27897220

  5. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    PubMed Central

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of the human degradome composed of 561 proteases and homologs. This increased complexity of rat proteases mainly derives from the expansion of several families, including placental cathepsins, testases, kallikreins, and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in rat and mouse and may contribute to explain some functional differences between these closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with mouse protease inhibitors and the expansion of families of cysteine and serine protease inhibitors in rodents with respect to human. These comparative analyses may provide new views on the functional diversity of proteases and inhibitors and contribute to the development of innovative strategies for treating proteolysis diseases. PMID:15060002

  6. The anticancer activity of the fungal metabolite terrecyclic acid A is associated with modulation of multiple cellular stress response pathways.

    PubMed

    Turbyville, Thomas J; Wijeratne, E M Kithsiri; Whitesell, Luke; Gunatilaka, A A Leslie

    2005-10-01

    Tumors are dependent on cellular stress responses, in particular the heat shock response, for survival in their hypoxic, acidotic, and nutrient-deprived microenvironments. Using cell-based reporter assays, we have identified terrecyclic acid A (TCA) from Aspergillus terreus, a fungus inhabiting the rhizosphere of Opuntia versicolor of the Sonoran desert, as a small-molecule inducer of the heat shock response that shows anticancer activity. Further characterization suggested that TCA also affects oxidative and inflammatory cellular stress response pathways. The presence of an alpha-methylene ketone moiety suggested that TCA may form adducts with sulfhydryl groups of proteins. Reaction with labile intracellular cysteines was supported by our finding that the glutathione precursor N-acetyl-cysteine protected tumor cells from the cytotoxic effects of TCA whereas the glutathione-depleting agent buthionine sulfoximine enhanced its activity. Related sesquiterpenes have been shown to increase levels of reactive oxygen species (ROS) and to inhibit nuclear factor kappaB (NF-kappaB) transcriptional activity. To assess whether TCA could have similar activities, we used a ROS-sensitive dye and flow cytometry to show that TCA does indeed increase ROS levels in 3LL cells. When tested in cells carrying NF-kappaB reporter constructs, TCA also exhibited concentration-dependent inhibition of cytokine-induced NF-kappaB transcriptional activity. These findings suggest that TCA modulates multiple stress pathways-the oxidative, heat shock, and inflammatory responses-in tumor cells that promote their survival. Small-molecule natural products such as TCA may serve as useful probes for understanding the relationships between these pathways, potentially providing leads for the design of novel and effective anticancer drugs.

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

    PubMed

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

    2013-10-01

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

  8. Molecular markers of serine protease evolution

    PubMed Central

    Krem, Maxwell M.; Di Cera, Enrico

    2001-01-01

    The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and α/β-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains. PMID:11406580

  9. 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.

  10. 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...

  11. Proteases in bacterial pathogenesis.

    PubMed

    Ingmer, Hanne; Brøndsted, Lone

    2009-11-01

    Bacterial pathogens rely on proteolysis for protein quality control under adverse conditions experienced in the host, as well as for the timely degradation of central virulence regulators. We have focused on the contribution of the conserved Lon, Clp, HtrA and FtsH proteases to pathogenesis and have highlighted common biological processes for which their activities are important for virulence.

  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-induced corrosion of wire rope

    SciTech Connect

    Little, B.; Ray, R.; Hart, K.; Wagner, P.

    1995-10-01

    Localized corrosion of carbon steel wire rope stored in a humid environment on wooden spools was caused by organic acid and carbon dioxide production by fungi growing directly on the wood. Fungal growth was found on the interior so the wooden spools, and corrosion was most severe on the wrap of wire in direct contact with the wood. Laboratory experiments and an extensive review of the literature demonstrated causal relationships between storage conditions and fungal growth and localized corrosion.

  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. 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).

  16. 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

  17. Proteases in blood clotting.

    PubMed

    Walsh, Peter N; Ahmad, Syed S

    2002-01-01

    The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

  18. Management of protease inhibitor-associated hyperlipidemia.

    PubMed

    Penzak, Scott R; Chuck, Susan K

    2002-01-01

    and lovastatin are significantly metabolized by cytochrome P450 enzymes (CYP3A4) and are therefore not recommended for coadministration with protease inhibitors. A fibric acid derivative (gemfibrozil or fenofibrate) should be used in patients with primary hypertriglyceridemia. However, it must be kept in mind that protease inhibitors, such as nelfinavir and ritonavir, induce enzymes involved in the metabolism of the fibric acid derivatives and may, therefore, reduce the lipid-lowering activity of coadministered gemfibrozil or fenofibrate. In certain patients HMG-CoA reductase inhibitors may be used in combination with fibric acid derivatives but patients should be carefully monitored for liver and skeletal muscle toxicity. Select patients may experience improvements in serum lipid levels when their offending protease inhibitor(s) is/are exchanged for efavirenz, nevirapine, or abacavir; however each patient's virologic and immunologic status must be taken closely into consideration.

  19. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations.

    PubMed

    Wang, Liping; Ridgway, Darin; Gu, Tingyue; Moo-Young, Murray

    2005-03-01

    Filamentous fungi have long been used for the production of metabolites and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have also achieved increased attention as hosts for recombinant DNA. However, the production levels of non-fungal proteins are usually low. Despite the achievements obtained using molecular tools, the heterologous protein loss caused by extracellular fungal protease degradation persists. This review provides an overview of the potential bioprocessing strategies that can be applied to inhibit protease activity thereby enhancing heterologous protein production.

  20. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    PubMed

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  1. 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.

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

    PubMed

    Renicke, Christian; Spadaccini, Roberta; Taxis, Christof

    2013-01-01

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

  3. 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.

  4. Association of circulating factor seven activating protease (FSAP) and of oral Omega-3 fatty acids supplements with clinical outcome in patients with atrial fibrillation: the OMEGA-AF study.

    PubMed

    Parahuleva, Mariana S; Kanse, Sandip; Hölschermann, Hans; Zheleva, Kirila; Zandt, Daniel; Worsch, Michael; Parviz, Behnoush; Güttler, Norbert; Tillmanns, Harald; Böning, Andreas; Erdogan, Ali

    2014-04-01

    Factor VII Activating Protease (FSAP) activates factor VII (FVII) as well as pro-urokinase (uPA). Our goal was to evaluate the relation between plasma levels of FSAP and clinical instability in atrial fibrillation (AF) and possible effects of oral omega-3 fatty acids (FA) supplements. 101 patients with persistent AF were analyzed in the OMEGA-AF Study. Plasma FSAP levels were measured at baseline and after 12 weeks of treatment with omega-3 FA. The median FSAP antigen concentration, in contrast to FSAP activity, was higher in patients with persistent AF. The maintenance of SR after successful cardioversion (CV) did not lead to a normalization of FSAP concentration. Supplementation with omega-3 FA but not placebo significantly reduced elevated FSAP concentration. Furthermore, elevated FSAP levels did not indicate a significantly increased risk of recurrence of AF after electrical CV or cardiovascular clinical events during 1 year of follow-up. Plasma FSAP concentration was increased in patients with AF and may be involved in the pathogenesis of this condition. The possible effects of omega-3 FA on clinical AF potential could be linked with modulation of circulating FSAP levels.

  5. In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects.

    PubMed Central

    Lech, W J; Wang, G; Yang, Y L; Chee, Y; Dorman, K; McCrae, D; Lazzeroni, L C; Erickson, J W; Sinsheimer, J S; Kaplan, A H

    1996-01-01

    We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity. PMID:8627733

  6. A new member of the plasma protease inhibitor gene family.

    PubMed Central

    Ragg, H

    1986-01-01

    A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II. Images PMID:3003690

  7. Universal fungal vaccines

    PubMed Central

    Hamad, Mawieh

    2012-01-01

    The complex nature of fungal pathogens, the intricate host-pathogen relationship and the health status of subjects in need of antifungal vaccination continue to hamper efforts to develop fungal vaccines for clinical use. That said, the rise of the universal vaccine concept is hoped to revive fungal vaccine research by expanding the pool of vaccine candidates worthy of clinical evaluation. It can do so through antigenic commonality-based screening for vaccine candidates from a wide range of pathogens and by reassessing the sizable collection of already available experimental and approved vaccines. Development of experimental vaccines protective against multiple fungal pathogens is evidence of the utility of this concept in fungal vaccine research. However, universal fungal vaccines are not without difficulties; for instance, development of vaccines with differential effectiveness is an issue that should be addressed. Additionally, rationalizing the development of universal fungal vaccines on health or economic basis could be contentious. Herein, universal fungal vaccines are discussed in terms of their potential usefulness and possible drawbacks. PMID:22922769

  8. 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.

  9. 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

  10. 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.

  11. Temporal dependence of cysteine protease activation following excitotoxic hippocampal injury.

    PubMed

    Berry, J N; Sharrett-Field, L J; Butler, T R; Prendergast, M A

    2012-10-11

    Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium (Ca(2+))-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity inhibits N-methyl-D-aspartate- (NMDA-) induced excitotoxic (20 μM NMDA) cell death and changes in synaptophysin immunoreactivity. Significant NMDA-induced cytotoxicity (as measured by propidium iodide [PI] uptake) was found in the CA1 region of the hippocampus at all timepoints examined (24, 72, 120 h), an effect significantly attenuated by co-exposure to the selective NMDA receptor antagonist DL-2-Amino-5-phosphonopentanoic acid (APV), but not MDL-28170, a potent cysteine protease inhibitor. Results indicated sparing of NMDA-induced loss of the synaptic vesicular protein synaptophysin in all regions of the hippocampus by MDL-28170, though only at early timepoints after injury. These results suggest Ca(2+)-dependent recruitment of cysteine proteases within 24h of excitotoxic insult, but activation of alternative cellular degrading mechanisms after 24h. Further, these data suggest that synaptophysin may be a substrate for calpains and related proteases.

  12. 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.

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

    PubMed Central

    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

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

    PubMed Central

    Quintero, David; Bermudes, David

    2014-01-01

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

  15. 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.

  16. Contribution of Aspartic Proteases in Candida Virulence. Protease Inhibitors against Candida Infections.

    PubMed

    Staniszewska, Monika; Małgorzata, Bondaryk; Zbigniew, Ochal

    2016-08-09

    Candida species are the major opportunistic human pathogens accounting for 70-90% of all invasive fungal infections. Candida spp, especially C. albicans, are able to produce and secrete hydrolytic enzymes, particularly aspartic proteases (Saps). These enzymes production is an evolutionary adaptation of pathogens to utilize nutrients and survive in host. Sap1-10 are believed to contribute to the adhesion and invasion of host tissues through the degradation of cell surface structures. Aspartic proteases control several steps in innate immune evasion and they degrade proteins related to immunological defense (antibodies, complement and cytokines), allowing the fungus to escape from the first line of host defense. The existing ways to identify potential drug targets rely on specific subset like virulence genes, transcriptional and stress response factors. Candida virulence factors like Sap isoenzymes can be pivotal targets for drug development. The identification of mechanism of a non-canonical inflammasome exerted by Saps could open novel therapeutic strategies to dampen hyperinflammatory response in candidiasis.

  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. Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

    PubMed Central

    Gharamah, Abdullah A; Moharram, Ahmed M; Ismail, Mady A; AL-Hussaini, Ashraf K

    2014-01-01

    Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2), sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin). Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss. PMID:24008795

  19. Cathepsin proteases in Toxoplasma gondii

    PubMed Central

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

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

  20. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    PubMed Central

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2014-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains. PMID:24359934

  1. 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

  2. Fungal arthritis and osteomyelitis.

    PubMed

    Kohli, Rakhi; Hadley, Susan

    2005-12-01

    Fungal arthritis and osteomyelitis are uncommon diseases and generally present in an indolent fashion. The incidence of fungal bone and joint dis-ease is increasing with an increase in the prevalence of factors predisposing to invasive fungal disease, such as the use of central venous catheters, broad spectrum antibiotics, immunosuppression, and abdominal surgery. Definitive diagnosis relies on bone or synovial culture or biopsy. Successful management has traditionally consisted of amphotericin B in combination with surgical debridement. Given the rarity of this disease, treatment is not well defined, but reports of success with the use of azole antifungal agents, including itraconazole, fluconazole, voriconazole, and posaconazole, are promising.

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

    PubMed Central

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

    2014-01-01

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

  4. 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

  5. Granuloma, fungal (Majocchi's) (image)

    MedlinePlus

    This is a picture of a fungal granuloma, a large, red (erythematous) patch (plaque) with a prominent border. Within the borders of the lesion are scattered blisters (pustules) that indicate deeper ...

  6. 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

  7. Lvserpin3 is involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

    PubMed

    Liu, Yongjie; Liu, Tao; Hou, Fujun; Wang, Xianzong; Liu, Xiaolin

    2016-01-01

    Serine protease inhibitor, represented by serpin, plays an important inhibitory role on proteases involved in the immune responses. To clarify the immune characterizations of serpin, a novel serpin (Lvserpin3) encoding for 410 amino acids with a 23-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Lvserpin3 expressed strongest in hepatopancreas, and was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Suppression of Lvserpin3 by dsRNA led to a significant increase in the transcripts of LvPPAF, LvproPO and phenoloxidase (PO) activity, and also led to the high cumulative mortality. The recombinant Lvserpin3 protein (rLvserpin3) inhibited the proteases secreted by M. lysodeikticus and Bacillus subtilis, and further exhibited inhibitory role on the growth of B. subtilis and M. lysodeikticu. Moreover, rLvserpin3 was found to be able to block the activation of prophenoloxidase system. Taken together, the results imply that Lvserpin3 may be involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

  8. Application of Protease Technology in Dermatology

    PubMed Central

    Del Rosso, James Q.

    2013-01-01

    This article reviews background on proteases and their functions, their physiological significance in skin, and the potential implications of incorporating specific proteases and protease blends into dermatological products, including skin care formulations. The history of protease blend formulations used in wound model studies and for other disorders is reviewed. In vitro data with use of a specific 3-protease blend with evaluation of the impact on various skin proteins and peptides is also discussed in this article. PMID:23882305

  9. Metabolism in Fungal Pathogenesis

    PubMed Central

    Ene, Iuliana V.; Brunke, Sascha; Brown, Alistair J.P.; Hube, Bernhard

    2014-01-01

    Fungal pathogens must assimilate local nutrients to establish an infection in their mammalian host. We focus on carbon, nitrogen, and micronutrient assimilation mechanisms, discussing how these influence host–fungus interactions during infection. We highlight several emerging trends based on the available data. First, the perturbation of carbon, nitrogen, or micronutrient assimilation attenuates fungal pathogenicity. Second, the contrasting evolutionary pressures exerted on facultative versus obligatory pathogens have led to contemporary pathogenic fungal species that display differing degrees of metabolic flexibility. The evolutionarily ancient metabolic pathways are conserved in most fungal pathogen, but interesting gaps exist in some species (e.g., Candida glabrata). Third, metabolic flexibility is generally essential for fungal pathogenicity, and in particular, for the adaptation to contrasting host microenvironments such as the gastrointestinal tract, mucosal surfaces, bloodstream, and internal organs. Fourth, this metabolic flexibility relies on complex regulatory networks, some of which are conserved across lineages, whereas others have undergone significant evolutionary rewiring. Fifth, metabolic adaptation affects fungal susceptibility to antifungal drugs and also presents exciting opportunities for the development of novel therapies. PMID:25190251

  10. Immunotherapy of Fungal Infections.

    PubMed

    Datta, Kausik; Hamad, Mawieh

    2015-01-01

    Fungal organisms are ubiquitous in the environment. Pathogenic fungi, although relatively few in the whole gamut of microbial pathogens, are able to cause disease with varying degrees of severity in individuals with normal or impaired immunity. The disease state is an outcome of the fungal pathogen's interactions with the host immunity, and therefore, it stands to reason that deep/invasive fungal diseases be amenable to immunotherapy. Therefore, antifungal immunotherapy continues to be attractive as an adjunct to the currently available antifungal chemotherapy options for a number of reasons, including the fact that existing antifungal drugs, albeit largely effective, are not without limitations, and that morbidity and mortality associated with invasive mycoses are still unacceptably high. For several decades, intense basic research efforts have been directed at development of fungal immunotherapies. Nevertheless, this approach suffers from a severe bench-bedside disconnect owing to several reasons: the chemical and biological peculiarities of the fungal antigens, the complexities of host-pathogen interactions, an under-appreciation of the fungal disease landscape, the requirement of considerable financial investment to bring these therapies to clinical use, as well as practical problems associated with immunizations. In this general, non-exhaustive review, we summarize the features of ongoing research efforts directed towards devising safe and effective immunotherapeutic options for mycotic diseases, encompassing work on antifungal vaccines, adoptive cell transfers, cytokines, antimicrobial peptides (AMPs), monoclonal antibodies (mAbs), and other agents.

  11. Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii.

    PubMed

    Yu, Gang; Liu, Jin-Liang; Xie, Li-Qin; Wang, Xue-Liang; Zhang, Shi-Hong; Pan, Hong-Yu

    2012-12-01

    The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

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

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

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

  13. The Evolution of Fungal Metabolic Pathways

    PubMed Central

    Rokas, Antonis

    2014-01-01

    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

  14. 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

  15. Association of a protease with polytene chromosomes of Drosophila melanogaster.

    PubMed

    Cavagnaro, J; Pierce, D A; Lucchesi, J C; Chae, C B

    1980-11-01

    Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques. The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.

  16. 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

  17. Proteochemometrics mapping of the interaction space for retroviral proteases and their substrates.

    PubMed

    Kontijevskis, Aleksejs; Petrovska, Ramona; Yahorava, Sviatlana; Komorowski, Jan; Wikberg, Jarl E S

    2009-07-15

    Understanding the complex interactions of retroviral proteases with their ligands is an important scientific challenge in efforts to achieve control of retroviral infections. Development of drug resistance because of high mutation rates and extensive polymorphisms causes major problems in treating the deadly diseases these viruses cause, and prompts efforts to identify new strategies. Here we report a comprehensive analysis of the interaction of 63 retroviral proteases from nine different viral species with their substrates and inhibitors based on publicly available data from the past 17years of retroviral research. By correlating physico-chemical descriptions of retroviral proteases and substrates to their biological activities we constructed a highly statistically valid 'proteochemometric' model for the interactome of retroviral proteases. Analysis of the model indicated amino acid positions in retroviral proteases with the highest influence on ligand activity and revealed general physicochemical properties essential for tight binding of substrates across multiple retroviral proteases. Hexapeptide inhibitors developed based on the discovered general properties effectively inhibited HIV-1 proteases in vitro, and some exhibited uniformly high inhibitory activity against all HIV-1 proteases mutants evaluated. A generalized proteochemometric model for retroviral proteases interactome has been created and analysed in this study. Our results demonstrate the feasibility of using the developed general strategy in the design of inhibitory peptides that can potentially serve as templates for drug resistance-improved HIV retardants.

  18. 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.

  19. 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.

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

    PubMed

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

    2013-03-01

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

  1. Botulinum neurotoxin A protease: discovery of natural product exosite inhibitors.

    PubMed

    Silhár, Peter; Capková, Katerina; Salzameda, Nicholas T; Barbieri, Joseph T; Hixon, Mark S; Janda, Kim D

    2010-03-10

    A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid (I1) is disclosed. A detailed evaluation of chicoric acid's mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination. Other components found in Echinacea, I3 and I4, were also inhibitors of the protease.

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

    PubMed

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

    2010-07-15

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

  3. Molecular docking and structure-based virtual screening studies of potential drug target, CAAX prenyl proteases, of Leishmania donovani.

    PubMed

    Singh, Shalini; Vijaya Prabhu, Sitrarasu; Suryanarayanan, Venkatesan; Bhardwaj, Ruchika; Singh, Sanjeev Kumar; Dubey, Vikash Kumar

    2016-11-01

    Targeting CAAX prenyl proteases of Leishmania donovani can be a good approach towards developing a drug molecule against Leishmaniasis. We have modeled the structure of CAAX prenyl protease I and II of L. donovani, using homology modeling approach. The structures were further validated using Ramachandran plot and ProSA. Active site prediction has shown difference in the amino acid residues present at the active site of CAAX prenyl protease I and CAAX prenyl protease II. The electrostatic potential surface of the CAAX prenyl protease I and II has revealed that CAAX prenyl protease I has more electropositive and electronegative potentials as compared CAAX prenyl protease II suggesting significant difference in their activity. Molecular docking with known bisubstrate analog inhibitors of protein farnesyl transferase and peptidyl (acyloxy) methyl ketones reveals significant binding of these molecules with CAAX prenyl protease I, but comparatively less binding with CAAX prenyl protease II. New and potent inhibitors were also found using structure-based virtual screening. The best docked compounds obtained from virtual screening were subjected to induced fit docking to get best docked configurations. Prediction of drug-like characteristics has revealed that the best docked compounds are in line with Lipinski's rule. Moreover, best docked protein-ligand complexes of CAAX prenyl protease I and II are found to be stable throughout 20 ns simulation. Overall, the study has identified potent drug molecules targeting CAAX prenyl protease I and II of L. donovani whose drug candidature can be verified further using biochemical and cellular studies.

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

    PubMed

    Theron, Louwrens W; Divol, Benoit

    2014-11-01

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

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

    PubMed

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

    2005-12-15

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

  6. Mast Cell Proteases and Inflammation

    PubMed Central

    Dai, Hongyan; Korthuis, Ronald J.

    2011-01-01

    Mast cells are best known for their role in allergic reactions but are also now recognized for their important contributions to a number of disparate inflammatory conditions through the release of inflammatory mediators, serglycin and other proteoglycans, and proteases. Because these tissue resident inflammatory cells express proteases in such great abundance and their enzymatic activity results in cleavage of a multitude of proteins and peptides, which in turn modify tissue function, their substrate specificity, tissue distribution, and mode of action have become the subjects of great interest. Although mast cell protease-dependent proteolysis is critical to host defense against invading pathogens, regulation of these hydrolytic enzymes is essential to limiting self-induced damage as well. Indeed, dysregulated release of mast cell proteases is now recognized to contribute to the pathogenesis of a number of inflammatory conditions including asthma, abdominal aortic aneurysm formation, vessel damage in atherosclerosis and hypertension, arthritis, and ischemia/reperfusion injury. Understanding how mast cell proteases contribute to inflammation will thus help unravel molecular mechanisms that underlie such immunologic disorders and will help identify new therapeutic targets for drug development. PMID:22125569

  7. 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.

  8. Active protease mapping in 2DE gels.

    PubMed

    Zhao, Zhenjun; Russell, Pamela J

    2009-01-01

    Proteases act as the molecular mediators of many vital biological processes. To understand the function of each protease, it needs to be separated from other proteins and characterized in its natural, biologically active form. In the method described in this chapter, proteases in a biological sample are separated under nonreducing conditions in 2DE gels. A specific small protease substrate, tagged with a fluorescent dye, is copolymerized into the SDS gel in the second dimension. After electrophoresis, the proteins are renatured by washing the gel with Triton X-100 solution or Milli Q water to remove SDS. The gel is then incubated in a protease assay buffer. The hydrolysis of the tagged specific substrate by the renatured protease releases the free fluorescent dye, which fluoresces in situ. The fluorescent spots indicate the location of the specific proteases in the gel and the specificity of the proteases.

  9. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  10. Fungal rhinitis in dogs.

    PubMed

    Ostrzeszewicz, M; Sapierzyński, R

    2015-01-01

    Fungal rhinitis and sinusitis in dogs are quite common reasons of chronic nasal discharge and rhinoscopy in such cases is commonly suggested. Forty three dogs were examined using rhinoscopy because of the presence of chronic airway symptoms. Clinical examination, routine hematology and serum biochemistry profiles, nasal and frontal sinus radiographs were made in all animals. Additionally, computed tomography in one dog was performed. Samples for histopathology were taken from 9 patients during rhinoscopy, additionally, from 8 of these patients samples for cytopathology were collected by blind nasal swab technique. In 9 of 43 dogs (20,5%), all males aged 1 to 13 years, examinations led to a diagnosis of fungal rhinitis. In 2 cases a diagnosis of fungal rhinitis was obtained based solely on cytopathology, while in 7 cases - mycosis of nasal mucosa was confirmed by histopathology. The present study revealed that cytopathological examination of nasal swabs has a low diagnostic value in the case of nasal infections in dogs. Although, in some dogs cytopathology, together with other widely available diagnostic techniques was sufficient to reliably diagnose fungal rhinitis, histopathology of samples collected during rhinoscopy is still the gold standard in such cases.

  11. Immunoregulation in Fungal Diseases

    PubMed Central

    Roussey, Jonathan A.; Olszewski, Michal A.; Osterholzer, John J.

    2016-01-01

    This review addresses specific regulatory mechanisms involved in the host immune response to fungal organisms. We focus on key cells and regulatory pathways involved in these responses, including a brief overview of their broader function preceding a discussion of their specific relevance to fungal disease. Important cell types discussed include dendritic cells and regulatory T cells, with a focus on specific studies relating to their effects on immune responses to fungi. We highlight the interleukin-10, programmed cell death 1, and cytotoxic T lymphocyte-associated protein 4 signaling pathways and emphasize interrelationships between these pathways and the regulatory functions of dendritic cells and regulatory T cells. Throughout our discussion, we identify selected studies best illustrating the role of these cells and pathways in response to specific fungal pathogens to provide a contextual understanding of the tightly-controlled network of regulatory mechanisms critical to determining the outcome of exposure to fungal pathogens. Lastly, we discuss two unique phenomena relating to immunoregulation, protective tolerance and immune reactivation inflammatory syndrome. These two clinically-relevant conditions provide perspective as to the range of immunoregulatory mechanisms active in response to fungi. PMID:27973396

  12. Fungal Burn Wound Infection

    DTIC Science & Technology

    1991-01-01

    severely limits the may prove to be useful in burn patients. Clotrimazole , applied clinical utility of such a culture. Biopsy and frozen-section and as...useful in wound and permit prompt institution of appropriate the treatment of systemic fungal infections. Clotrimazole is treatment. poorly absorbed

  13. Sputum fungal smear

    MedlinePlus

    ... A.D.A.M. Editorial team. Related MedlinePlus Health Topics Fungal Infections Lung Diseases Browse the Encyclopedia A.D.A.M., Inc. is accredited by URAC, also known as the American Accreditation HealthCare ... for online health information and services. Learn more about A.D. ...

  14. 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 ...

  15. Nail Fungal Infections

    MedlinePlus

    ... Are treatments usually effective?Are there any side effects of the treatment?If my treatment works, will my nail grow back normally?If I've had one fungal nail infection, am I likely to get another?What kinds of shoes should I wear?Should I wear gloves when ...

  16. 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

  17. Plant cysteine proteases that evoke itch activate protease-activated receptors

    PubMed Central

    Reddy, V.B.; Lerner, E.A.

    2013-01-01

    Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

  18. 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.

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

    PubMed Central

    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

  20. New perspectives towards analysing fungal communities in terrestrial environments

    PubMed

    Kowalchuk

    1999-06-01

    Fungi play key roles in numerous ecosystem functions, and recent advances in the study of fungal diversity and ecology have led to a greater appreciation of this group of microeukaryotes. The application of a variety of nucleic acid techniques to fungal classification and phylogeny has led to a number of evolutionary insights, and has also begun to provide the necessary information for identification of unknown isolates and DNA sequences. These data, together with direct molecular characterisation of fungi in the field, provide new possibilities to describe fungal diversity and distribution. Such advances will no doubt also provide the means for a more detailed interpretation of ecological experiments.

  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. The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters*

    PubMed Central

    Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

    2015-01-01

    Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

  4. 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...

  5. Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

    PubMed

    Arasappan, A; Njoroge, F G; Chan, T-Y; Bennett, F; Bogen, S L; Chen, K; Gu, H; Hong, L; Jao, E; Liu, Y-T; Lovey, R G; Parekh, T; Pike, R E; Pinto, P; Santhanam, B; Venkatraman, S; Vaccaro, H; Wang, H; Yang, X; Zhu, Z; Mckittrick, B; Saksena, A K; Girijavallabhan, V; Pichardo, J; Butkiewicz, N; Ingram, R; Malcolm, B; Prongay, A; Yao, N; Marten, B; Madison, V; Kemp, S; Levy, O; Lim-Wilby, M; Tamura, S; Ganguly, A K

    2005-10-01

    We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

  6. Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation.

    PubMed

    Hu, Junjie; Liu, Fei; Ju, Huangxian

    2015-04-21

    A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

  7. Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica.

    PubMed Central

    Gonzalez-Lopez, Claudia I; Szabo, Roman; Blanchin-Roland, Sylvie; Gaillardin, Claude

    2002-01-01

    Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway. PMID:11861549

  8. 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

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

    PubMed

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

    1987-07-01

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

  10. Proteases of Stored Product Insects and Their Inhibition by Specific Protease Inhibitors from Soybeans and Wheat Grain

    DTIC Science & Technology

    1989-12-15

    PROTEASES; PROTEASE INHIBITORS; STORED-PRODUCT INISECTS; TRIBOLIUM CASIANEUH; MIDGUT PROTEASES; TENEBRIO MOLITOR MIDGUT-PROTEASES; LOCUST CAECAL...separation and identification of numerous midgut proteases in Tenebrio and Tribolium . The PAGE-gelatin matrix revealed the inhibitory effect of BBI...the proteinaceous trypsin-chymotrypsin inhibitor from soybeans) on several Tribolium proteases - an effect which was not detectable in inhibition

  11. Structure of the NS2B-NS3 protease from Zika virus after self-cleavage

    PubMed Central

    Phoo, Wint Wint; Li, Yan; Zhang, Zhenzhen; Lee, Michelle Yueqi; Loh, Ying Ru; Tan, Yaw Bia; Ng, Elizabeth Yihui; Lescar, Julien; Kang, CongBao; Luo, Dahai

    2016-01-01

    The recent outbreak of Zika virus (ZIKV) infections in the Americas represents a serious threat to the global public health. The viral protease that processes viral polyproteins during infection appears as an attractive drug target. Here we report a crystal structure at 1.84 Å resolution of ZIKV non-structural protein NS2B-NS3 protease with the last four amino acids of the NS2B cofactor bound at the NS3 active site. This structure represents a post-proteolysis state of the enzyme during viral polyprotein processing and provides insights into peptide substrate recognition by the protease. Nuclear magnetic resonance (NMR) studies and protease activity assays unravel the protein dynamics upon binding the protease inhibitor BPTI in solution and confirm this finding. The structural and functional insights of the ZIKV protease presented here should advance our current understanding of flavivirus replication and accelerate structure-based antiviral drug discovery against ZIKV. PMID:27845325

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

    PubMed

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

    2016-09-02

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

  13. 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.

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

    PubMed

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

    2010-06-15

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

  15. Degradation of Argininosuccinate Lyase by a Protease Synthesized in Soybean Cell Suspension Cultures 1

    PubMed Central

    Shargool, P. D.

    1975-01-01

    Suspension cultures of soybean (Glycine max L.) were shown to contain protease activity which could be inhibited by the addition of protease inhibitors such as p-hydroxymercuribenzoate and ethylenediaminetetraacetic acid. The use of these inhibitors, coupled with studies of the rate of degradation of argininosuccinate lyase (argininosuccinate-lyase = l-arginino-succinate arginine-lyase, EC 4.3.2.1) in extracts of cell cultures grown for 24 hours led to the hypothesis that a metal-dependent protease is synthesized by the cells after 24 hours of growth, to remove the lyase enzyme. PMID:16659138

  16. Bacterial proteases in IBD and IBS.

    PubMed

    Steck, Natalie; Mueller, Kerstin; Schemann, Michael; Haller, Dirk

    2012-11-01

    Proteases play a decisive role in health and disease. They fulfil diverse functions and have been associated with the pathology of gastrointestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). The current knowledge focuses on host-derived proteases including matrix metalloproteinases, various serine proteases and cathepsins. The possible contribution of bacterial proteases has been largely ignored in the pathogenesis of IBD and IBS, although there is increasing evidence, especially demonstrated for proteases from pathogenic bacteria. The underlying mechanisms extend to proteases from commensal bacteria which may be relevant for disease susceptibility. The intestinal microbiota and its proteolytic capacity exhibit the potential to contribute to the pathogenesis of IBD and IBS. This review highlights the relevance of host- and bacteria-derived proteases and their signalling mechanisms.

  17. 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

  18. Occult Fungal Scleritis

    PubMed Central

    Jeang, Lauren J.; Davis, Aaron; Madow, Brian; Espana, Edgar M.; Margo, Curtis E.

    2017-01-01

    Purpose To heighten awareness of occult fungal scleritis. Method Case report and review of the literature. Results A 73-year-old woman with diabetes mellitus was diagnosed for 3 months with immune-mediated scleritis and subsequently treated with corticosteroids. On referral, the patient had a scleral nodule with contiguous corneal infiltrate and hypopyon. Culture grew Fusarium species not further classified. The infection could not be controlled with antifungal therapy, and the eye was removed. No exogenous or endogenous source for the infection could be identified by clinical history or examination. Conclusion Fungal scleritis can develop in persons without a history of foreign body injury, minor trauma, or evidence of endogenous fungemia. A high index of suspicion for infectious scleritis must be maintained in persons with presumed immune-mediated scleritis who fail to respond to conventional therapy, particularly if they present with decreased visual acuity. PMID:28275602

  19. Unusual fungal niches.

    PubMed

    Cantrell, S A; Dianese, J C; Fell, J; Gunde-Cimerman, N; Zalar, P

    2011-01-01

    Fungi are found in all aerobic ecosystems, colonizing a diversity of substrates and performing a wide diversity of functions, some of which are not well understood. Many spices of fungi are cosmopolitan and generalists or habitats. Unusual fungal niches are habitats where extreme conditions would be expected to prevent the development of a mycobiota. In this review we describe five unusual fungal habitats in which fungi occupy poorly understood niches: Antarctic dry valleys, high Arctic glaciers, salt flats and salterns, hypersaline microbial mats and plant trichomes. Yeasts, black yeast-like fungi, melanized filamentous species as well as representatives of Aspergillus and Penicillium seem to be dominant among the mycobiota adapted to cold and saline niches. Plant trichomes appear to be a taxa. The advent of new sequencing technologies is helping to elucidate the microbial diversity in many ecosystems, but more studies are needed to document the functional role of fungi in the microbial communities thriving in these unusual environments.

  20. 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.

  1. Proteins as nitrogen source for plants: a short story about exudation of proteases by plant roots.

    PubMed

    Adamczyk, Bartosz; Smolander, Aino; Kitunen, Veikko; Godlewski, Mirosław

    2010-07-01

    Interest in the problem of plant nitrogen nutrition is increasing. Certain plants can use not only inorganic nitrogen, but also intact amino acids and short peptides. According to our studies, the roots of several agricultural and wild-living plants are able to exude proteases and by using them to create a pool of accessible N. This mini-review offers an overview of the problem of protease exudation by plant roots and its potential role in plant nitrogen nutrition.

  2. Fungal CSL transcription factors

    PubMed Central

    Převorovský, Martin; Půta, František; Folk, Petr

    2007-01-01

    Background The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. Results We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. Conclusion Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans. PMID:17629904

  3. 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

  4. 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.

  5. Design of new potent HTLV-1 protease inhibitors: in silico study.

    PubMed

    Kheirabadi, Mitra; Maleki, Javad; Soufian, Safieh; Hosseini, Samaneh

    2016-03-01

    HTLV-1 and HIV-1 are two major causes for severe T-cell leukemia disease and acquired immune deficiency syndrome (AIDS). HTLV-1 protease, a member of aspartic acid protease family, plays important roles in maturation during virus replication cycle. The impairment of these proteases results in uninfectious HTLV-1virions.Similar to HIV-1protease deliberate mutations that confer drug resistance on HTLV-1 are frequently seen in this protease. Therefore, inhibition of HTLV-1 protease activity is expected to disrupt HTLV-1's ability to replicate and infect additional cells. In this study, we initially designed fifteen inhibitory compounds based on the conformations of a class of HIV-1 aspartyl protease inhibitors, sulfonamid-peptoid. Five compounds were chosen based on the goodness of their Drug-Likeness scoreusing "Lipinsk's rule of five". Here, using protein-ligand docking approach we compared the inhibitory constants of these compounds to those available in literatures and observed significantly higher inhibition for two compounds, SP-4 and SP-5. Our data suggest that the addition of two cyclic hydrocarbons to both ends of sulfonamide peptoids leads to the formation of new hydrophobic interactions due to the semi-circular form of these compounds, connecting the first chain of protease to the two ends of tested ligands via Hydrophobic interactions. We conclude that hydrophobic force plays an important role in suppressing protease activity especially for HTLV-1 protease, which in turn prevents the virus maturity. Therefore, designing and development of new ligands based on aromatic hydrocarbons in both ends of inhibitors is very promising for efficient treatment.

  6. Design of new potent HTLV-1 protease inhibitors: in silico study

    PubMed Central

    Kheirabadi, Mitra; Maleki, Javad; Soufian, Safieh; Hosseini, Samaneh

    2016-01-01

    HTLV-1 and HIV-1 are two major causes for severe T-cell leukemia disease and acquired immune deficiency syndrome (AIDS). HTLV-1 protease, a member of aspartic acid protease family, plays important roles in maturation during virus replication cycle. The impairment of these proteases results in uninfectious HTLV-1virions.Similar to HIV-1protease deliberate mutations that confer drug resistance on HTLV-1 are frequently seen in this protease. Therefore, inhibition of HTLV-1 protease activity is expected to disrupt HTLV-1’s ability to replicate and infect additional cells. In this study, we initially designed fifteen inhibitory compounds based on the conformations of a class of HIV-1 aspartyl protease inhibitors, sulfonamid-peptoid. Five compounds were chosen based on the goodness of their Drug-Likeness scoreusing “Lipinsk’s rule of five”. Here, using protein-ligand docking approach we compared the inhibitory constants of these compounds to those available in literatures and observed significantly higher inhibition for two compounds, SP-4 and SP-5. Our data suggest that the addition of two cyclic hydrocarbons to both ends of sulfonamide peptoids leads to the formation of new hydrophobic interactions due to the semi-circular form of these compounds, connecting the first chain of protease to the two ends of tested ligands via Hydrophobic interactions. We conclude that hydrophobic force plays an important role in suppressing protease activity especially for HTLV-1 protease, which in turn prevents the virus maturity. Therefore, designing and development of new ligands based on aromatic hydrocarbons in both ends of inhibitors is very promising for efficient treatment. PMID:27844017

  7. Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets.

    PubMed

    Coupe, Simon A; Sinclair, Ben K; Watson, Lyn M; Heyes, Julian A; Eason, Jocelyn R

    2003-03-01

    Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 degrees C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO(2), 5% O(2)) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73-89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

  8. A novel serine protease inhibitor from Bungarus fasciatus venom.

    PubMed

    Lu, Jia; Yang, Hailong; Yu, Haining; Gao, Weikai; Lai, Ren; Liu, Jingze; Liang, Xingcai

    2008-03-01

    By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

  9. 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.)

  10. 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.

  11. Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity.

    PubMed

    Wongsuk, Thanwa; Pumeesat, Potjaman; Luplertlop, Natthanej

    2016-05-01

    When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell-cell communication, microorganisms secrete molecules called quorum-sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast-to-hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

  12. PROSPER: An Integrated Feature-Based Tool for Predicting Protease Substrate Cleavage Sites

    PubMed Central

    Perry, Andrew J.; Akutsu, Tatsuya; Webb, Geoffrey I.; Whisstock, James C.; Pike, Robert N.

    2012-01-01

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

  13. Isolation of pyrrolocins A-C: cis- and trans-decalin tetramic acid antibiotics from an endophytic fungal-derived pathway.

    PubMed

    Jadulco, Raquel C; Koch, Michael; Kakule, Thomas B; Schmidt, Eric W; Orendt, Anita; He, Haiyin; Janso, Jeffrey E; Carter, Guy T; Larson, Erica C; Pond, Christopher; Matainaho, Teatulohi K; Barrows, Louis R

    2014-11-26

    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.

  14. 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

  15. Nelfinavir: fourth protease inhibitor approved.

    PubMed

    1997-01-01

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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. Fungal pretreatment of lignocellulosic biomass.

    PubMed

    Wan, Caixia; Li, Yebo

    2012-01-01

    Pretreatment is a crucial step in the conversion of lignocellulosic biomass to fermentable sugars and biofuels. Compared to thermal/chemical pretreatment, fungal pretreatment reduces the recalcitrance of lignocellulosic biomass by lignin-degrading microorganisms and thus potentially provides an environmentally-friendly and energy-efficient pretreatment technology for biofuel production. This paper provides an overview of the current state of fungal pretreatment by white rot fungi for biofuel production. The specific topics discussed are: 1) enzymes involved in biodegradation during the fungal pretreatment; 2) operating parameters governing performance of the fungal pretreatment; 3) the effect of fungal pretreatment on enzymatic hydrolysis and ethanol production; 4) efforts for improving enzymatic hydrolysis and ethanol production through combinations of fungal pretreatment and physical/chemical pretreatment; 5) the treatment of lignocellulosic biomass with lignin-degrading enzymes isolated from fungal pretreatment, with a comparison to fungal pretreatment; 6) modeling, reactor design, and scale-up of solid state fungal pretreatment; and 7) the limitations and future perspective of this technology.

  1. 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.

  2. Effect of protease EPg222 obtained from Penicillium chrysogenum isolated from dry-cured ham in pieces of pork loins.

    PubMed

    Benito, María J; Rodríguez, Mar; Sosa, María J; Martín, Alberto; Córdoba, Juan J

    2003-01-01

    The fungal protease EPg222 obtained from Penicillium chrysogenum Pg222 isolated from dry-cured ham, was assayed for proteolytic activity in a meat model system based on sterile pieces of pork loins for 32 days. Treated samples showed a significative reduction of total high ionic strength-soluble proteins during the incubation period, as compared with a control incubated without enzyme, both on the surface and in the depth. SDS-PAGE analysis of this protein fraction showed higher hydrolysis of the main myofibrillar proteins H-meromyosin, actin, and tropomyosin in treated samples. Non-protein and amino acidic nitrogen were detected in higher amounts in enzyme-added than in control pieces of loins, both on the surface and in the depth. Thus, addition of enzyme EPg222 to whole pieces of meat results in an increase of protein hydrolysis. The effect of this enzyme could be of great interest for stimulating proteolysis in whole dry-cured meat pieces.

  3. Structure and mechanism of rhomboid protease.

    PubMed

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

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

  4. Allergic Fungal Sinusitis.

    PubMed

    Correll, Daniel P; Luzi, Scott A; Nelson, Brenda L

    2015-12-01

    A 42 year old male presents with worsening pain and an increase in thick chronic drainage of the left sinus. Image studies show complete opacification of the left frontal sinus, left sphenoid sinus, and the left maxillary sinus. The patient was taken to the operating room and tissue for microscopic evaluation was obtained. The microscopic findings were classic for allergic fungal sinusitis: areas of alternating mucinous material and inflammatory cell debris and abundant Charcot-Leyden crystals. Cultures were performed and the patient began steroid therapy and desensitization therapy.

  5. Peptide inhibitors of Dengue virus NS3 protease. Part 1: Warhead.

    PubMed

    Yin, Zheng; Patel, Sejal J; Wang, Wei-Ling; Wang, Gang; Chan, Wai-Ling; Rao, K R Ranga; Alam, Jenefer; Jeyaraj, Duraiswamy A; Ngew, Xinyi; Patel, Viral; Beer, David; Lim, Siew Pheng; Vasudevan, Subhash G; Keller, Thomas H

    2006-01-01

    Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.

  6. Bacterial proteases: targets for diagnostics and therapy.

    PubMed

    Kaman, W E; Hays, J P; Endtz, H P; Bikker, F J

    2014-07-01

    Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

  7. 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.

  8. 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.

  9. Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients

    PubMed Central

    Kempf, Dale J.; Isaacson, Jeffrey D.; King, Martin S.; Brun, Scott C.; Xu, Yi; Real, Kathryn; Bernstein, Barry M.; Japour, Anthony J.; Sun, Eugene; Rode, Richard A.

    2001-01-01

    The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with reduced susceptibility. Mutations at positions 82, 54, 10, 63, 71, and 84 were most closely associated with relatively modest (4- and 10-fold) changes in phenotype, while the K20M/R and F53L mutations, in conjunction with multiple other mutations, were associated with >20- and >40-fold-reduced susceptibility, respectively. The median 50% inhibitory concentrations (IC50) of lopinavir against isolates with 0 to 3, 4 or 5, 6 or 7, and 8 to 10 of the above 11 mutations were 0.8-, 2.7-, 13.5-, and 44.0-fold higher, respectively, than the IC50 against wild-type HIV. On average, the IC50 of lopinavir increased by 1.74-fold per mutation in isolates containing three or more mutations. Each of the 16 viruses that displayed a >20-fold change in susceptibility contained mutations at residues 10, 54, 63, and 82 and/or 84, along with a median of three mutations at residues 20, 24, 46, 53, 71, and 90. The number of protease mutations from the 11 identified in these analyses (the lopinavir mutation score) may be useful for the interpretation of HIV genotypic resistance testing with respect to lopinavir-ritonavir (Kaletra) regimens and may provide insight into the genetic barrier to resistance to lopinavir-ritonavir in both antiretroviral therapy-naive and protease inhibitor-experienced patients. PMID:11462018

  10. 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.

  11. 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.

  12. Production and purification of novel thermostable alkaline protease from Anoxybacillus sp. KP1.

    PubMed

    Matpan Bekler, F; Acer, Ö; Güven, K

    2015-09-26

    In this study, an extracellular novel alkaline protease (EC 3.4.21-24, 99) from a thermophilic and aerobic strain of Anoxybacillus sp. KP1 has been studied. Maximum protease activity was obtained at 50 degC at pH 9.0 after 24 hours of incubation. Among the carbon and nitrogen sources used; the optimum protease production was with soluble starch, maltose, urea and casamino acid. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel chromatography. Molecular weight of purified enzyme was determined as 106 kDa by SDS-PAGE. Purified protease was stable at 50-60 °C and at pH 9.0 for 1 h. The enzyme activity was increased in the presence of Ca2+, Cu2+, Tween 80 and Triton X-100, however the enzyme activity was inhibited in the presence of Hg2+, ethylene diamine tetra acetic acid (EDTA) and H2O2. Proteolytic activity was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF). The enzyme seems to be a serine alkaline protease. In the presence of detergents, the protease was clearly stable and residual activity was between 73-82%.

  13. 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.

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

    PubMed

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

    2007-01-01

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

  15. Purification and characterization of aspartic protease derived from Sf9 insect cells.

    PubMed

    Gotoh, Takeshi; Ono, Hiroki; Kikuchi, Ken-Ichi; Nirasawa, Satoru; Takahashi, Saori

    2010-01-01

    An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 µM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) µM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.

  16. 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.

  17. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

    PubMed

    Renatus, Martin; Parrado, Shirley Gil; D'Arcy, Allan; Eidhoff, Ulf; Gerhartz, Bernd; Hassiepen, Ulrich; Pierrat, Benoit; Riedl, Ralph; Vinzenz, Daniela; Worpenberg, Susanne; Kroemer, Markus

    2006-08-01

    Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

  18. Molecular Determinants of MecA as a Degradation Tag for the ClpCP Protease*

    PubMed Central

    Mei, Ziqing; Wang, Feng; Qi, Yutao; Zhou, Zhiyuan; Hu, Qi; Li, Han; Wu, Jiawei; Shi, Yigong

    2009-01-01

    Regulated proteolysis by ATP-dependent proteases is universal in all living cells. In Bacillus subtilis, the degradation of the competence transcription factor ComK is mediated by a ternary complex involving the adaptor protein MecA and the ATP-dependent protease ClpCP. Here we demonstrate that a C-terminal, 98-amino acid domain of MecA (residues 121–218) serves as a non-recycling, degradation tag and targets a variety of fusion proteins to the ClpCP protease for degradation. MecA-(121–218) facilitates productive oligomerization of ClpC, stimulates the ATPase activity of ClpC, and allows the activated ClpC complex to stably associate with ClpP. Importantly, the ClpCP protease undergoes dynamic cycles of assembly and disassembly, which are triggered by association with MecA and the degradation of MecA, respectively. PMID:19767395

  19. Modulation of an apparent mRNA pool for extracellular protease in Bacillus amyloliquefaciens.

    PubMed Central

    O'Connor, R; Elliott, W H; May, B K

    1978-01-01

    Late-log-phase cells of Bacillus amyloliquefaciens have the unusual capacity to produce extracellular protease for over 60 min in the presence of rifampin or actinomycin D at levels which strongly inhibit incorporation of amino acids into cellular protein. If cells are incubated in the presence of high levels of amino acids for 75 min this capacity is exhausted, but it is retained if the incubation is carried out in low levels of amino acids. Transfer of exhausted cells from high to low concentrations of amino acids results in a progressive recovery of the capacity for rifampin-actinomycin-insensitive protease production. The results seem best explained on the basis of the accumulation of a reserve pool of mRNA for extracellular protease. Measurement of the apparent mRNA pool size over 12 h shows a cyclical rise and fall, and these changes correlate with a periodic variation of the rate of protease production. A working hypothesis is presented to account for these observations in terms of a novel control situation over protease mRNA transcription. PMID:711667

  20. Serious fungal infections in Pakistan.

    PubMed

    Jabeen, K; Farooqi, J; Mirza, S; Denning, D; Zafar, A

    2017-02-04

    The true burden of fungal infection in Pakistan is unknown. High-risk populations for fungal infections [tuberculosis (TB), diabetes, chronic respiratory diseases, asthma, cancer, transplant and human immunodeficiency virus (HIV) infection] are numerous. Here, we estimate the burden of fungal infections to highlight their public health significance. Whole and at-risk population estimates were obtained from the WHO (TB), BREATHE study (COPD), UNAIDS (HIV), GLOBOCAN (cancer) and Heartfile (diabetes). Published data from Pakistan reporting fungal infections rates in general and specific populations were reviewed and used when applicable. Estimates were made for the whole population or specific populations at risk, as previously described in the LIFE methodology. Of the 184,500,000 people in Pakistan, an estimated 3,280,549 (1.78%) are affected by a serious fungal infection, omitting all cutaneous infection, oral candidiasis and allergic fungal sinusitis, which we could not estimate. Compared with other countries, the rates of candidaemia (21/100,000) and mucormycosis (14/100,000) are estimated to be very high, and are based on data from India. Chronic pulmonary aspergillosis rates are estimated to be high (39/100,000) because of the high TB burden. Invasive aspergillosis was estimated to be around 5.9/100,000. Fungal keratitis is also problematic in Pakistan, with an estimated rate of 44/100,000. Pakistan probably has a high rate of certain life- or sight-threatening fungal infections.

  1. 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.

  2. A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides.

    PubMed

    Thekkiniath, Jose C; Zabet-Moghaddam, Masoud; San Francisco, Susan K; San Francisco, Michael J

    2013-06-01

    Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.

  3. Fungal infections in immunocompromised travelers.

    PubMed

    Lortholary, Olivier; Charlier, Caroline; Lebeaux, David; Lecuit, Marc; Consigny, Paul Henri

    2013-03-01

    Immunocompromised patients represent an increasing group of travelers, for business, tourism, and visiting friends and relatives. Those with severe cellular immunodeficiency (advanced human immunodeficiency virus infection and transplant recipients) display the highest risk of fungal infections. International travel is less risky in most other types of immunodeficiency (except those with neutropenia). A systematic visit in a travel clinic for immunocompromised patients traveling to the tropics ensures that the specific risks of acquiring fungal infections (and others) are understood. When immunocompromised hosts return to their area of residence, a nonbacteriologically documented, potentially severe, febrile pneumonia, with or without dissemination signs (skin lesions, cytopenia) should alert for travel-acquired fungal infection, even years after return. Localized subcutaneous nodule may be also ascribed to fungal infection. Finally, infectious diseases physicians should be aware of major clinical patterns of travel-acquired fungal infection, as well as the fungi involved, and risk factors according to the geographical area visited.

  4. Invertebrate models of fungal infection.

    PubMed

    Arvanitis, Marios; Glavis-Bloom, Justin; Mylonakis, Eleftherios

    2013-09-01

    The morbidity, mortality and economic burden associated with fungal infections, together with the emergence of fungal strains resistant to current antimicrobial agents, necessitate broadening our understanding of fungal pathogenesis and discovering new agents to treat these infections. Using invertebrate hosts, especially the nematode Caenorhabditis elegans and the model insects Drosophila melanogaster and Galleria mellonella, could help achieve these goals. The evolutionary conservation of several aspects of the innate immune response between invertebrates and mammals makes the use of these simple hosts an effective and fast screening method for identifying fungal virulence factors and testing potential antifungal compounds. The purpose of this review is to compare several model hosts that have been used in experimental mycology to-date and to describe their different characteristics and contribution to the study of fungal virulence and the detection of compounds with antifungal properties. This article is part of a Special Issue entitled: Animal Models of Disease.

  5. [Genetic diagnosis against fungal cerebromeningitis].

    PubMed

    Ohno, Hideaki; Miyazaki, Yoshitsugu

    2013-01-01

    Fungal cerebromeningitis is one of deep seated mycoses and also a fatal fungal infectious disease. Regarding to causative pathogen of fungal cerebromeningitis in Japan, Cryptococcus spp., Candida spp., Aspergillus spp., are popular fungi. In general, the diagnosis of deep seated mycosis is sometime difficult. The genetic diagnosis method such as PCR against deep seated mycosis has been developing and it has been also reported as one of useful diagnostic tests. However, PCR for fungal detection is still a research test that has not been cleared or approved officially, therefore it should not be used for diagnosis, or patient management routinely. The PCR which detect broad range of fungi or specific fungus is applied for clinical situation, a careful attention should be paid for avoiding contamination because many fungal species are available in living environment.

  6. Evaluation of Pyrolysis Oil as Carbon Source for Fungal Fermentation

    PubMed Central

    Dörsam, Stefan; Kirchhoff, Jennifer; Bigalke, Michael; Dahmen, Nicolaus; Syldatk, Christoph; Ochsenreither, Katrin

    2016-01-01

    Pyrolysis oil, a complex mixture of several organic compounds, produced during flash pyrolysis of organic lignocellulosic material was evaluated for its suitability as alternative carbon source for fungal growth and fermentation processes. Therefore several fungi from all phyla were screened for their tolerance toward pyrolysis oil. Additionally Aspergillus oryzae and Rhizopus delemar, both established organic acid producers, were chosen as model organisms to investigate the suitability of pyrolysis oil as carbon source in fungal production processes. It was observed that A. oryzae tolerates pyrolysis oil concentrations between 1 and 2% depending on growth phase or stationary production phase, respectively. To investigate possible reasons for the low tolerance level, eleven substances from pyrolysis oil including aldehydes, organic acids, small organic compounds and phenolic substances were selected and maximum concentrations still allowing growth and organic acid production were determined. Furthermore, effects of substances to malic acid production were analyzed and compounds were categorized regarding their properties in three groups of toxicity. To validate the results, further tests were also performed with R. delemar. For the first time it could be shown that small amounts of phenolic substances are beneficial for organic acid production and A. oryzae might be able to degrade isoeugenol. Regarding pyrolysis oil toxicity, 2-cyclopenten-1-on was identified as the most toxic compound for filamentous fungi; a substance never described for anti-fungal or any other toxic properties before and possibly responsible for the low fungal tolerance levels toward pyrolysis oil. PMID:28066378

  7. Evaluation of Pyrolysis Oil as Carbon Source for Fungal Fermentation.

    PubMed

    Dörsam, Stefan; Kirchhoff, Jennifer; Bigalke, Michael; Dahmen, Nicolaus; Syldatk, Christoph; Ochsenreither, Katrin

    2016-01-01

    Pyrolysis oil, a complex mixture of several organic compounds, produced during flash pyrolysis of organic lignocellulosic material was evaluated for its suitability as alternative carbon source for fungal growth and fermentation processes. Therefore several fungi from all phyla were screened for their tolerance toward pyrolysis oil. Additionally Aspergillus oryzae and Rhizopus delemar, both established organic acid producers, were chosen as model organisms to investigate the suitability of pyrolysis oil as carbon source in fungal production processes. It was observed that A. oryzae tolerates pyrolysis oil concentrations between 1 and 2% depending on growth phase or stationary production phase, respectively. To investigate possible reasons for the low tolerance level, eleven substances from pyrolysis oil including aldehydes, organic acids, small organic compounds and phenolic substances were selected and maximum concentrations still allowing growth and organic acid production were determined. Furthermore, effects of substances to malic acid production were analyzed and compounds were categorized regarding their properties in three groups of toxicity. To validate the results, further tests were also performed with R. delemar. For the first time it could be shown that small amounts of phenolic substances are beneficial for organic acid production and A. oryzae might be able to degrade isoeugenol. Regarding pyrolysis oil toxicity, 2-cyclopenten-1-on was identified as the most toxic compound for filamentous fungi; a substance never described for anti-fungal or any other toxic properties before and possibly responsible for the low fungal tolerance levels toward pyrolysis oil.

  8. 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.

  9. 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

  10. Biological roles of fungal carotenoids.

    PubMed

    Avalos, Javier; Carmen Limón, M

    2015-08-01

    Carotenoids are terpenoid pigments widespread in nature, produced by bacteria, fungi, algae and plants. They are also found in animals, which usually obtain them through the diet. Carotenoids in plants provide striking yellow, orange or red colors to fruits and flowers, and play important metabolic and physiological functions, especially relevant in photosynthesis. Their functions are less clear in non-photosynthetic microorganisms. Different fungi produce diverse carotenoids, but the mutants unable to produce them do not exhibit phenotypic alterations in the laboratory, apart of lack of pigmentation. This review summarizes the current knowledge on the functional basis for carotenoid production in fungi. Different lines of evidence support a protective role of carotenoids against oxidative stress and exposure to visible light or UV irradiation. In addition, the carotenoids are intermediary products in the biosynthesis of physiologically active apocarotenoids or derived compounds. This is the case of retinal, obtained from the symmetrical oxidative cleavage of β-carotene. Retinal is the light-absorbing prosthetic group of the rhodopsins, membrane-bound photoreceptors present also in many fungal species. In Mucorales, β-carotene is an intermediary in the synthesis of trisporoids, apocarotenoid derivatives that include the sexual hormones the trisporic acids, and they are also presumably used in the synthesis of sporopollenin polymers. In conclusion, fungi have adapted their ability to produce carotenoids for different non-essential functions, related with stress tolerance or with the synthesis of physiologically active by-products.

  11. Bacterial proteases from the intracellular vacuole niche; protease conservation and adaptation for pathogenic advantage.

    PubMed

    Huston, Wilhelmina M

    2010-06-01

    Proteases with important roles for bacterial pathogens that specifically reside within intracellular vacuoles are frequently homologous to those that have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialized functions for intracellular vacuole-residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria that remain to be described. This review summarizes the recent findings of novel protease functions from the intracellular human pathogenic bacteria that reside exclusively in vacuoles.

  12. Continuous Proteolysis with a stabilized stabilized protease. I. Chemical stabilization of an alkaline protease.

    PubMed

    Boudrant, J; Cuq, J L; Cheftel, C

    1976-12-01

    Due to the loss of enzymatic activity as a function of time, an alkaline protease, selected for the continuous preparation of protein hydrolysates (J. Boudrant and C. Cheftel, Biotechnol. Bioeng., 18,1735, 1976), was chemically stabilized by a simple treatment with glutaraldehyde. Two fractions, soluble and insoluble, were obtained. The activities of these two fractions were measured with casein and N-benzoyl-L-arginine ethyl ester (BAEE) as a function of glutaraldehyde concentration used. It was noted that the insoluble fraction was practically inactive with the first substrate and that the heat stability of the soluble form was likewise enhanced. Molecular weights of these two forms were unchanged, but the uv-spectrum of the soluble form was modified. From amino acid analysis, it appears that this treatment mainly provokes a decrease in lysine content.

  13. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor.

    PubMed Central

    Ho, D D; Toyoshima, T; Mo, H; Kempf, D J; Norbeck, D; Chen, C M; Wideburg, N E; Burt, S K; Erickson, J W; Singh, M K

    1994-01-01

    Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease. Images PMID:8107264

  14. Dual detection of fungal infections in Drosophila through recognition of microbial structures and sensing of virulence factors

    PubMed Central

    Gottar, Marie; Gobert, Vanessa; Matskevich, Alexey A.; Reichhart, Jean-Marc; Wang, Chengshu; Butt, Tariq M.; Belvin, Marcia; Hoffmann, Jules A.; Ferrandon, Dominique

    2007-01-01

    The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the Pattern Recognition Receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. Here, we report that GNBP3 is a novel Pattern Recognition Receptor that is required for the detection of fungal cell wall components. Strikingly, we find that there is a second, parallel, pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host. PMID:17190605

  15. Superficial fungal infections.

    PubMed

    Schwartz, Robert A

    Superficial fungal infections arise from a pathogen that is restricted to the stratum corneum, with little or no tissue reaction. In this Seminar, three types of infection will be covered: tinea versicolor, piedra, and tinea nigra. Tinea versicolor is common worldwide and is caused by Malassezia spp, which are human saprophytes that sometimes switch from yeast to pathogenic mycelial form. Malassezia furfur, Malassezia globosa, and Malassezia sympodialis are most closely linked to tinea versicolor. White and black piedra are both common in tropical regions of the world; white piedra is also endemic in temperate climates. Black piedra is caused by Piedraia hortae; white piedra is due to pathogenic species of the Trichosporon genus. Tinea nigra is also common in tropical areas and has been confused with melanoma.

  16. Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.

    PubMed

    Czurda, S; Smelik, S; Preuner-Stix, S; Nogueira, F; Lion, T

    2016-01-01

    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.

  17. 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

  18. 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.

  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. 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.

  1. Serious fungal infections in Korea.

    PubMed

    Huh, K; Ha, Y E; Denning, D W; Peck, K R

    2017-02-04

    Information on the incidence and prevalence of fungal infections is of critical value in public health policy. However, nationwide epidemiological data on fungal infections are scarce, due to a lack of surveillance and funding. The objective of this study was to estimate the disease burden of fungal infections in the Republic of Korea. An actuarial approach using a deterministic model was used for the estimation. Data on the number of populations at risk and the frequencies of fungal infections in those populations were obtained from national statistics reports and epidemiology papers. Approximately 1 million people were estimated to be affected by fungal infections every year. The burdens of candidemia (4.12 per 100,000), cryptococcal meningitis (0.09 per 100,000), and Pneumocystis pneumonia (0.51 per 100,000) in South Korea were estimated to be comparable to those in other countries. The prevalence of chronic pulmonary aspergillosis (22.4 per 100,000) was markedly high, probably due to the high burden of tuberculosis in Korea. The low burdens of allergic bronchopulmonary aspergillosis (56.9 per 100,000) and severe asthma with fungal sensitization (75.1 per 100,000) warrant further study. Oral candidiasis (539 per 100,000) was estimated to affect a much larger population than noted in previous studies. Our work provides valuable insight on the epidemiology of fungal infections; however, additional studies are needed.

  2. 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

  3. Serious fungal infections in Portugal.

    PubMed

    Sabino, R; Verissímo, C; Brandão, J; Martins, C; Alves, D; Pais, C; Denning, D W

    2017-02-10

    There is a lack of knowledge on the epidemiology of fungal infections worldwide because there are no reporting obligations. The aim of this study was to estimate the burden of fungal disease in Portugal as part of a global fungal burden project. Most published epidemiology papers reporting fungal infection rates from Portugal were identified. Where no data existed, specific populations at risk and fungal infection frequencies in those populations were used in order to estimate national incidence or prevalence, depending on the condition. An estimated 1,510,391 persons develop a skin or nail fungal infection each year. The second most common fungal infection in Portugal is recurrent vulvovaginal candidiasis, with an estimated 150,700 women (15-50 years of age) suffering from it every year. In human immunodeficiency virus (HIV)-infected people, oral or oesophageal candidiasis rates were estimated to be 19.5 and 16.8/100,000, respectively. Candidaemia affects 2.19/100,000 patients, in a total of 231 cases nationally. Invasive aspergillosis is less common than in other countries as chronic obstructive pulmonary disease (COPD) is uncommon in Portugal, a total of 240 cases annually. The estimated prevalence of chronic pulmonary aspergillosis after tuberculosis (TB) is 194 cases, whereas its prevalence for all underlying pulmonary conditions was 776 patients. Asthma is common (10% in adults) and we estimate 16,614 and 12,600 people with severe asthma with fungal sensitisation and allergic bronchopulmonary aspergillosis, respectively. Sixty-five patients develop Pneumocystis pneumonia in acquired immune deficiency syndrome (AIDS) and 13 develop cryptococcosis. Overall, we estimate a total number of 1,695,514 fungal infections starting each year in Portugal.

  4. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS).

    PubMed

    Boulware, Kevin T; Daugherty, Patrick S

    2006-05-16

    We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

  5. 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

  6. Enzymatic nanolithography of FRET peptide layer using V8 protease-immobilized AFM probe.

    PubMed

    Nakamura, Chikashi; Miyamoto, Chie; Obataya, Ikuo; Takeda, Seiji; Yabuta, Masayuki; Miyake, Jun

    2007-04-15

    In our study, a method based on Enzymatic nanolithography was successfully performed in a buffered solution using Staphylococcal serine V8 protease and AFM. To estimate the lithographing activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed fluorescence resonance energy transfer (FRET) peptides as reporter peptides that showed enzymatic action specific to the V8 protease. When the protease digested the reporter peptide a quencher residue was released from the peptide and resulted in the appearance of fluorescence. In the designed 9-mer peptides, TAMRA functioned as a good quencher for FAM. When the fluorescence resonance energy transfer peptides immobilized on a glass substrate were hydrolyzed by V8 protease at the C-terminal of glutamic acid, fluorescence of a reporter dye was observed because of the release of a quencher from the substrate. After contacting and lateral scanning of the protease-immobilized AFM tip to the reporter peptide layer, a fluorescent area was observed by imaging using total internal refection fluorescence microscopy (TIRFM). The increment of fluorescence intensity of the digested peptide indicates the performance of lithography. Lithographing rates increased in inverse relation to scanning rates of the probe. The maximum limit of the scanning rate, i.e., that was too fast to permit cutting of the peptide on the substrate, and the lithographing performance are discussed in this study.

  7. An extremely thermotolerant, alkaliphilic subtilisin-like protease from hyperthermophilic Bacillus sp. MLA64.

    PubMed

    Lagzian, Milad; Asoodeh, Ahmad

    2012-12-01

    The current work is a report on a new extremely thermostable protease from newly isolated hyperthermophilic Bacillus sp. MLA64. The protease was purified with a 16.5-fold increase in specific activity and 93.5% recovery. The molecular weight of the enzyme was estimated to be 24 kDa. The enzyme was extremely stable and quite active over the temperature range from 40 to 100°C with an optimal temperature at 95°C as well as in a wide range of pH from 6.0 to 12.5, with a superlative at pH 9.5. The enzyme activity was not enhanced in the presence of CaCl(2), indicating that the enzyme is calcium-independent. The enzyme showed high stability towards non-ionic surfactants and anionic surfactant SDS. In addition, the enzyme was relatively stable with respect to oxidizing agents. The protease was inhibited by PMSF but not by TPCK and TLCK, suggesting that it can be a subtilisin-like protease. Moreover, the N-terminal sequencing of the first 20 amino acids of the purified protease showed less homology with other well-known bacterial peptidases. In conclusion, the enzyme can be considered as a novel protease which might be a candidate for industrial processes.

  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. 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...

  10. The Role of Proteases and Exotoxin A in the Pathogenicity of Pseudomonas Aeruginosa Infections

    DTIC Science & Technology

    1981-01-01

    958. 1974. malinized sheep erythrocytes treated with tannic acid 3. Bjorn, M. I., Pavlovskis. 0. R.. Thompson. M. R. & and coated with protease or...using formalinized sheep erythrocytes 9:540, 1974. treated with tannic acid and coated with exotoxin of 61. Pavlovskis, 0. R.. Voelker. F. A...ing the views of the U.S. Navy D~epartment or the U.S. serologically distinct and have different amino acid naval service at large. composition and

  11. Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

    PubMed Central

    Lee, Sang Eun; Jeong, Se Kyoo

    2010-01-01

    Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD. PMID:20879045

  12. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis.

    PubMed

    Lee, Sang Eun; Jeong, Se Kyoo; Lee, Seung Hun

    2010-11-01

    Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/ PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.

  13. 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.

  14. 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.

  15. Production of a protease inhibitor from edible mushroom Agaricus bisporus and its statistical optimization by response surface methodology.

    PubMed

    Vishvakarma, Reena; Mishra, Abha

    2017-01-31

    The production of a protease inhibitor from Agaricus bisporus through solid state fermentation was studied. The purpose was to produce protease inhibitor from natural, cheap and readily available carbon and nitrogen sources. Solid state fermentation enhanced the mycelia growth and also gave a higher yield of the product. Further, fungal growth and other production parameters were statistically optimized. The specificity of the inhibitor was tested and was effective against trypsin. Screening of significant factors (wheat bran, cyanobacterial biomass, initial pH, temperature, incubation period, and moisture content and inoculum size) was done using Plackett-Burman Design. Central Composite Design was used to determine the optimized values of the significant variables which were found to be temperature (27.5 °C), incubation time (156 hrs.), cyanobacterial biomass (1 g) and moisture content (50%) and gave a statistical yield of 980 PIU/g which was 25.6% higher than experimental yield (780 PIU/g). The inhibitor was purified by ammonium sulphate precipitation and DEAE cellulose chromatography (yield 43.89% and 0.21% respectively) and subjected to Reversed-phase HPLC to validate its identity. Since protease inhibitors act against proteases, finding ample therapeutic roles; the isolated protease inhibitor from A. bisporus can also be a probable medicinal agent after its further characterization.

  16. Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle.

    PubMed

    Dias, B A; Neves, P M O J; Furlaneto-Maia, L; Furlaneto, M C

    2008-04-01

    A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.

  17. Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

    PubMed Central

    Zo, Young-Gun; Choe, Youngchool; Kim, Seon-Hee; Desai, Prashant V.; Avery, Mitchell A.; Craik, Charles S.; Kim, Tong-Soo; Rosenthal, Philip J.; Kong, Yoon

    2010-01-01

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

  18. 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

  19. 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.

  20. 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

  1. Fungal infections: a growing threat.

    PubMed Central

    Dixon, D M; McNeil, M M; Cohen, M L; Gellin, B G; La Montagne, J R

    1996-01-01

    THE EMERGENCE OF newly identified fungal pathogens and the reemergence of previously uncommon fungal diseases is primarily related to increases in the numbers of susceptible persons: people with HIV infection, bone marrow and organ transplant recipients, cancer patients being treated with chemotherapy, critically ill persons, and very low birth weight ( < or = 1500 g) infants. These immunocompromised populations are at risk for infection not only with opportunistic pathogens (for example, Pneumocystis, Candida, Cryptococcus, Trichosporon, Malassezia, Aspergillus, Penicillium marneffei, and numerous other moulds or yeasts) but also with fungal pathogens that usually infect otherwise healthy persons not previously exposed to endemic fungi (for example, Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis) and Sporothrix schenckii. Morbidity, mortality, and health care costs associated with fungal infections are high. Addressing the emergence of fungal diseases will require increased surveillance coupled with the availability of rapid, noninvasive diagnostic tests; monitoring the development of resistance to antifungal agents; and research focused on the understanding, prevention, and control of fungal infections. Images p[227]-a p226-a p232-a PMID:8643813

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing

    PubMed Central

    Mustafa, Ghulam; Arshad, Muhammad

    2017-01-01

    Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings. PMID:28367444

  7. Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing.

    PubMed

    Ahmed, Sibtain; Mustafa, Ghulam; Arshad, Muhammad; Rajoka, Muhammad Ibrahim

    2017-01-01

    Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings.

  8. Anthranilate-Activating Modules from Fungal Nonribosomal Peptide Assembly Lines†

    PubMed Central

    Ames, Brian D.; Walsh, Christopher T.

    2010-01-01

    Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted β-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities. PMID:20225828

  9. 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

  10. Role of cockroach proteases in allergic disease.

    PubMed

    Page, Kristen

    2012-10-01

    Allergic asthma is on the rise in developed countries, and cockroach exposure is a major risk factor for the development of asthma. In recent years, a number of studies have investigated the importance of allergen-associated proteases in modulating allergic airway inflammation. Many of the studies have suggested the importance of allergen-associated proteases as having a direct role on airway epithelial cells and dendritic cells. In most cases, activation of the protease activated receptor (PAR)-2 has been implicated as a mechanism behind the potent allergenicity associated with cockroaches. In this review, we focus on recent evidence linking cockroach proteases to activation of a variety of cells important in allergic airway inflammation and the role of PAR-2 in this process. We will highlight recent data exploring the potential mechanisms involved in the biological effects of the allergen.

  11. Serious fungal infections in Chile.

    PubMed

    Alvarez Duarte, E; Denning, D W

    2017-02-10

    The incidence and prevalence of fungal infections in Chile are unknown. Here, we have estimated the burden of serious fungal diseases from data obtained from clinical reports, WHO reports, Chilean census, OECD reports and comprehensive literature search available on PubMed and SciELO, among other scientific resources. Due the lack of official data about fungal diseases, frequencies were calculated based on the specific populations at risk. Recurrent vulvovaginal candidiasis (>4 episodes/year) is estimated to occur in 3108/100,000. Using a low international average rate of 5/100,000, we estimate 878 candidaemia cases and 132 patients with intra-abdominal candidiasis. Due to the low incidence of pulmonary tuberculosis (TB) in Chile, limited numbers of patients with chronic pulmonary aspergillosis are likely: a total of 1212, 25% following TB. Invasive aspergillosis is estimated to affect 296 patients following leukaemia therapy, transplantation and chronic obstructive pulmonary disease (COPD), 1.7/100,000. In addition, allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitisation (SAFS) were estimated to be around 97.9/100,000 and 127/100,000 respectively, in 675,772 adult asthmatics and 1700 CF patients. Given a 38,000 human immunodeficiency virus (HIV) population, with around 2189 new cases of acquired immune deficiency syndrome (AIDS) annually, cryptococcal meningitis and Pneumocystis pneumonia are estimated at 0.12/100,000 and 4.3/100,000, respectively. In total, 325,000 (1.9%) people in Chile develop serious fungal infections annually. Respiratory fungal disease predominates in Chile; a national action plan for fungal disease is urgently needed, including epidemiological studies to validate the estimates.

  12. Serious fungal infections in Ecuador.

    PubMed

    Zurita, J; Denning, D W; Paz-Y-Miño, A; Solís, M B; Arias, L M

    2017-02-04

    There is a dearth of data from Ecuador on the burden of life-threatening fungal disease entities; therefore, we estimated the burden of serious fungal infections in Ecuador based on the populations at risk and available epidemiological databases and publications. A full literature search was done to identify all epidemiology papers reporting fungal infection rates. WHO, ONU-AIDS, Index Mundi, Global Asthma Report, Globocan, and national data [Instituto Nacional de Estadística y Censos (INEC), Ministerio de Salud Pública (MSP), Sociedad de Lucha Contra el Cáncer (SOLCA), Instituto Nacional de Donación y Trasplante de Órganos, Tejidos y Células (INDOT)] were reviewed. When no data existed, risk populations were used to estimate frequencies of fungal infections, using previously described methodology by LIFE. Ecuador has a variety of climates from the cold of the Andes through temperate to humid hot weather at the coast and in the Amazon basin. Ecuador has a population of 15,223,680 people and an average life expectancy of 76 years. The median estimate of the human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) population at risk for fungal disease (<200 CD4 cell counts) is ∼10,000, with a rate of 11.1% (1100) of histoplasma, 7% (700) of cryptococcal meningitis, and 11% (1070) of Pneumocystis pneumonia. The burden of candidemia is 1037. Recurrent Candida vaginitis (≥4 episodes per year) affects 307,593 women aged 15-50 years. Chronic pulmonary aspergillosis probably affects ∼476 patients following tuberculosis (TB). Invasive aspergillosis is estimated to affect 748 patients (∼5.5/100,000). In addition, allergic bronchopulmonary aspergillosis (ABPA) in asthma and severe asthma with fungal sensitization (SAFS) were estimated to affect 26,642 and 45,013 people, respectively. Our estimates indicate that 433,856 (3%) of the population in Ecuador is affected by serious fungal infection.

  13. 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.

  14. 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.

  15. Protease Mediated Anti-Cancer Therapy

    DTIC Science & Technology

    2006-08-01

    anticancer therapy and focal light illumination is expected to be an effective treatment with reduced phototoxicity given the quenched state of the...to months following photodynamic therapy (PDT). Herein, we report a novel design of protease-mediated photosensitization by which phototoxicity can...W81XWH-05-1-0515 TITLE: Protease Mediated Anti-Cancer Therapy PRINCIPAL INVESTIGATOR: Ching-Hsuan Tung CONTRACTING ORGANIZATION

  16. PCSK9: an enigmatic protease.

    PubMed

    Lopez, Dayami

    2008-04-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge.

  17. 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

  18. Serious fungal infections in Canada.

    PubMed

    Dufresne, S F; Cole, D C; Denning, D W; Sheppard, D C

    2017-02-04

    There are currently no nationwide epidemiological data on fungal infections in Canada. We estimated the burden of serious fungal diseases using literature review and modeling, as per a methodology previously described by the LIFE program ( http://www.LIFE-worldwide.org ). Among the population of Canada (35.5 million in 2014), it was estimated that approximately 1.8% are affected by a serious fungal infection. Recurrent vulvovaginal candidiasis, severe asthma with fungal sensitization, and allergic bronchopulmonary aspergillosis are the most frequent infections, with population prevalences of 498,688 (1403/100,000), 73,344 (206/100,000), and 61,854 (174/100,000) cases, respectively. Over 3000 invasive fungal infections are estimated to occur annually, with incidences of 2068 cases (5.8/100,000) of invasive candidiasis, 566 cases (1.6/100,000) of invasive aspergillosis, 252 cases (0.71/100,000) of Pneumocystis pneumonia, 99 cases (0.28/100,000) of endemic mycoses, and 63 cases (0.18/100,000) of cryptococcosis. These estimates warrant validation through more formal epidemiological studies in Canada.

  19. Serious fungal infections in Peru.

    PubMed

    Bustamante, B; Denning, D W; Campos, P E

    2017-02-10

    Epidemiological data about mycotic diseases are limited in Peru and estimation of the fungal burden has not been previously attempted. Data were obtained from the Peruvian National Institute of Statistics and Informatics, UNAIDS and from the Ministry of Health's publications. We also searched the bibliography for Peruvian data on mycotic diseases, asthma, COPD, cancer and transplants. Incidence or prevalence for each fungal disease were estimated in specific populations at risk. The Peruvian population for 2015 was 31,151,543. In 2014, the estimated number of HIV/AIDS and pulmonary tuberculosis cases was 88,625, 38,581 of them not on ART, and 22,027, respectively. A total of 581,174 cases of fungal diseases were estimated, representing approximately 1.9% of the Peruvian population. This figure includes 498,606, 17,361 and 4,431 vulvovaginal, oral and esophageal candidiasis, respectively, 1,557 candidemia cases, 3,593 CPA, 1,621 invasive aspergillosis, 22,453 allergic bronchopulmonary aspergilllosis, 29,638 severe asthma with fungal sensitization, and 1,447 Pneumocystis pneumonia. This first attempt to assess the fungal burden in Peru needs to be refined. We believe the figure obtained is an underestimation, because of under diagnosis, non-mandatory reporting and lack of a surveillance system and of good data about the size of populations at risk.

  20. [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.

  1. An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

    PubMed Central

    Melnick, Laurence; Yang, Shiow-Shong; Rossi, Rick; Zepp, Charlie; Heefner, Donald

    1998-01-01

    We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity. PMID:9835523

  2. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

    PubMed

    Neveu, Julie; Regeard, Christophe; DuBow, Michael S

    2011-08-01

    The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

  3. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  4. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

    PubMed Central

    Ahmad Mazian, Mu'adz; Salleh, Abu Bakar; Basri, Mahiran; Rahman, Raja Noor Zaliha Raja Abd.

    2014-01-01

    Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. PMID:25093119

  5. Impact of aeration and agitation on metabolic heat and protease secretion of Aspergillus tamarii in a real-time biological reaction calorimeter.

    PubMed

    Dhandapani, Balaji; Mahadevan, Surianarayanan; Dhilipkumar, Sathish Sundar; Rajkumar, Suseela; Mandal, Asit Baran

    2012-06-01

    The effects of aeration and agitation on metabolic heat, alkaline protease production and morphology for Aspergillus tamarii MTCC5152 are reported in this manuscript. Measurement of metabolic heat has been attempted by the continuous and dynamic heat balance method in a biological real-time reaction calorimeter. At lower agitation intensities, growth-related processes were dominating. As a result the protease activity and the product heat yields were lower than those for 350 and 450 rpm. Although biomass growth was necessary to obtain maximum protease yield, agitation seemed to play a vital role in the protease production process. Energy dissipation per circulation function of the process is also deduced from power input. At optimal conditions, 350 rpm and 1 vvm, the gassed power required was 0.133 W. Pellet morphology and protease production were studied under different aeration and agitation intensities of A. tamarii. Pellet structure was considerably influenced by DO, a higher DO level resulted in denser pellets (1,018.4 kg/m(3)) leading to higher protease activity. Coupling of hydrodynamics and bio-reaction highlighted the complex relationship between energy dissipation, substrate uptake rate and fungal physiology. This study emphasised the potential of biocalorimetry as a reliable monitoring and robust control tool for aerobic fermentation of A. tamarii, using agricultural by-products.

  6. Protease expression by microorganisms and its relevance to crucial physiological/pathological events

    PubMed Central

    dos Santos, André Luis Souza

    2011-01-01

    The treatment of infections caused by fungi and trypanosomatids is difficult due to the eukaryotic nature of these microbial cells, which are similar in several biochemical and genetic aspects to host cells. Aggravating this scenario, very few antifungal and anti-trypanosomatidal agents are in clinical use and, therefore, therapy is limited by drug safety considerations and their narrow spectrum of activity, efficacy and resistance. The search for new bioactive agents against fungi and trypanosomatids has been expanded because progress in biochemistry and molecular biology has led to a better understanding of important and essential pathways in these microorganisms including nutrition, growth, proliferation, signaling, differentiation and death. In this context, proteolytic enzymes produced by these eukaryotic microorganisms are appointed and, in some cases, proven to be excellent targets for searching novel natural and/or synthetic pharmacological compounds, in order to cure or prevent invasive fungal/trypanosomatid diseases. With this task in mind, our research group and others have focused on aspartic-type proteases, since the activity of this class of hydrolytic enzymes is directly implicated in several facets of basic biological processes of both fungal and trypanosomatid cells as well as due to the participation in numerous events of interaction between these microorganisms and host structures. In the present paper, a concise revision of the beneficial effects of aspartic protease inhibitors, with emphasis on the aspartic protease inhibitors used in the anti-human immunodeficiency virus therapy, will be presented and discussed using our experience with the following microbial models: the yeast Candida albicans, the filamentous fungus Fonsecaea pedrosoi and the protozoan trypanosomatid Leishmania amazonensis. PMID:21537490

  7. Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi.

    PubMed

    Mucha, Joanna; Dahm, Hanna; Werner, Antoni

    2007-07-01

    The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

  8. Ecology of Fungal Plant Pathogens.

    PubMed

    Termorshuizen, Aad J

    2016-12-01

    Fungal plant pathogens are ubiquitous and highly diverse. Key to their success is high host density, which notably is the case in agroecosystems. Several hypotheses related to the effects of plant pathogens on plant diversity (the Janzen-Connell hypothesis, the dilution effect hypothesis) and the phenomenon of higher biomass in plant mixtures (i.e., overyielding) can all be explained by the quantitative interplay between host and pathogen density. In many agroecosystems, fungal plant pathogens cause great losses, since in monocultures diseased plants cannot be replaced by healthy plants. On the other hand, in natural ecosystems fungal plant pathogens shape the succession of vegetation and enhance the biodiversity of forests and grasslands. When pathogens are introduced into areas outside their natural range, they may behave differently, causing severe damage. Once introduced, changes may occur such as hybridization with other closely related pathogens or host shifts, host jumps, or horizontal gene transfer. Such changes can be hazardous for both agricultural and natural ecosystems.

  9. Four cats with fungal rhinitis.

    PubMed

    Whitney, Beth L; Broussard, John; Stefanacci, Joseph D

    2005-02-01

    Fungal rhinitis is uncommon in the cat and cases of nasal aspergillosis-penicilliosis have been rarely reported. Signs of fungal rhinitis include epistaxis, sneezing, mucopurulent nasal discharge and exophthalmos. Brachycephalic feline breeds seem to be at increased risk for development of nasal aspergillosis-penicilliosis. Computed tomography (CT) imaging and rhinoscopy are useful in assessing the extent of the disease and in obtaining diagnostic samples. Fungal culture may lead to false negative or positive results and must be used in conjunction with other diagnostic tests. Serological testing was not useful in two cats tested. The cats in this study were treated with oral itraconazole therapy. When itraconazole therapy was discontinued prematurely, clinical signs recurred. Hepatotoxicosis is a possible sequel to itraconazole therapy.

  10. Fungal infection following renal transplantation.

    PubMed

    Gallis, H A; Berman, R A; Cate, T R; Hamilton, J D; Gunnells, J C; Stickel, D L

    1975-09-01

    Twenty-seven deep fungal infections developed in 22 of 171 patients following renal transplantation. These infections included cryptococcosis (ten), nocardiosis (seven), candidiasis (four), aspergillosis (two), phycomycosis (two), chromomycosis (one), and subcutaneous infection with Phialophora gougeroti (one). Twelve infections occurred in living-related and ten in cadaveric recipients. Nineteen of the 22 patients were male. Infections occurred from 0 to 61 months after transplantation. Complicating non-fungal infections were present concomitantly in 15 patients. Thirteen patients died, eight probably as a result of fungal infection. Appropriate diagnostic procedures yielded a diagnosis in 20 of 27 infections, and therapy was begun in 18 patients. Serologic, culture, and biopsy procedures useful in making rapid diagnoses are advocated in the hope of increasing survival.

  11. The Fungal Defensin Family Enlarged

    PubMed Central

    Wu, Jiajia; Gao, Bin; Zhu, Shunyi

    2014-01-01

    Fungi are an emerging source of peptide antibiotics. With the availability of a large number of model fungal genome sequences, we can expect that more and more fungal defensin-like peptides (fDLPs) will be discovered by sequence similarity search. Here, we report a total of 69 new fDLPs encoded by 63 genes, in which a group of fDLPs derived from dermatophytes are defined as a new family (fDEF8) according to sequence and phylogenetic analyses. In the oleaginous fungus Mortierella alpine, fDLPs have undergone extensive gene expansion. Our work further enlarges the fungal defensin family and will help characterize new peptide antibiotics with therapeutic potential. PMID:25230677

  12. Metabolomics reveals insect metabolic responses associated with fungal infection.

    PubMed

    Xu, Yong-Jiang; Luo, Feifei; Gao, Qiang; Shang, Yanfang; Wang, Chengshu

    2015-06-01

    The interactions between insects and pathogenic fungi are complex. We employed metabolomic techniques to profile insect metabolic dynamics upon infection by the pathogenic fungus Beauveria bassiana. Silkworm larvae were infected with fungal spores and microscopic observations demonstrated that the exhaustion of insect hemocytes was coupled with fungal propagation in the insect body cavity. Metabolomic analyses revealed that fungal infection could significantly alter insect energy and nutrient metabolisms as well as the immune defense responses, including the upregulation of carbohydrates, amino acids, fatty acids, and lipids, but the downregulation of eicosanoids and amines. The insect antifeedant effect of the fungal infection was evident with the reduced level of maclurin (a component of mulberry leaves) in infected insects but elevated accumulations in control insects. Insecticidal and cytotoxic mycotoxins like oosporein and beauveriolides were also detected in insects at the later stages of infection. Taken together, the metabolomics data suggest that insect immune responses are energy-cost reactions and the strategies of nutrient deprivation, inhibition of host immune responses, and toxin production would be jointly employed by the fungus to kill insects. The data obtained in this study will facilitate future functional studies of genes and pathways associated with insect-fungus interactions.

  13. Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, meta...

  14. Rational reprogramming of fungal polyketide first-ring cyclization

    PubMed Central

    Xu, Yuquan; Zhou, Tong; Zhou, Zhengfu; Su, Shiyou; Roberts, Sue A.; Montfort, William R.; Zeng, Jia; Chen, Ming; Zhang, Wei; Lin, Min; Zhan, Jixun; Molnár, István

    2013-01-01

    Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides. PMID:23509261

  15. Intervention for hyperlipidemia associated with protease inhibitors.

    PubMed

    Melroe, N H; Kopaczewski, J; Henry, K; Huebsch, J

    1999-01-01

    In the past 3 years, treatment for HIV infection has significantly improved the prognosis for HIV-infected persons. The administration of protease inhibitors for the treatment of HIV infection has had a significant role in the reduction of AIDS-related complications. Recent findings have indicated that protease inhibitors may significantly increase lipids to levels that pose a health risk that may be greater than the illness itself. This article reviews the initial findings of a study that investigated the impact of interventions for the treatment of protease inhibitor-related hyperlipidemia. The purpose of the study was to determine if initiation of interventions based on the National Cholesterol Education Program Guidelines would be effective in lowering protease inhibitor-related hyperlipidemia without disrupting the effectiveness of the HIV therapy. A total of 45 HIV-infected individuals who were taking a protease inhibitor and had abnormally elevated lipids were enrolled into this study. Mean serum cholesterol level prior to initiation of a protease inhibitor regimen was 170 mg/dl as compared to a mean cholesterol at time of enrollment of 289 mg/dl and triglycerides of 879 mg/dl. Interventions included diet and exercise and the prescription of gemfibrozil alone or in combination with atorvatstatin. During the course of the study, overall intervention significantly reduced serum cholesterol level to 201 mg/dl (p. 01) over a study period of ten months. Case studies of five medical events related to hyperlipidemia are included. Currently, 26 participants continue in the study. Sixteen participants discontinued protease inhibitor therapy during the course of the study and thus ended their participation.

  16. Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

    PubMed

    Buch, Franziska; Kaman, Wendy E; Bikker, Floris J; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

  17. Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants

    PubMed Central

    Buch, Franziska; Kaman, Wendy E.; Bikker, Floris J.; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory. PMID:25750992

  18. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

    PubMed Central

    Loeb, D D; Hutchison, C A; Edgell, M H; Farmerie, W G; Swanstrom, R

    1989-01-01

    Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites. Images PMID:2642305

  19. Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant class IV chitinases have a small, amino-terminal chitin binding domain and a larger chitinase domain. Previous work on Zea mays chitinases ChitA and ChitB showed that their chitin binding domains bind insoluble chitin, that their catalytic domains degrade short, soluble forms of chitin, and th...

  20. Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37

    PubMed Central

    Bennici, Carmelo; Quatrini, Paola; Catania, Valentina; Mazzola, Salvatore; Ghersi, Giulio; Cuttitta, Angela

    2015-01-01

    Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended. PMID:26162075

  1. Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production.

    PubMed

    Outchkourov, Nikolay S; Rogelj, Boris; Strukelj, Borut; Jongsma, Maarten A

    2003-09-01

    Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.

  2. The QSAR and docking calculations of fullerene derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Saleh, Noha A.

    2015-02-01

    The inhibition of HIV-1 protease is considered as one of the most important targets for drug design and the deactivation of HIV-1. In the present work, the fullerene surface (C60) is modified by adding oxygen atoms as well as hydroxymethylcarbonyl (HMC) groups to form 6 investigated fullerene derivative compounds. These compounds have one, two, three, four or five O atoms + HMC groups at different positions on phenyl ring. The effect of the repeating of these groups on the ability of suggested compounds to inhibit the HIV protease is studied by calculating both Quantitative Structure Activity Relationship (QSAR) properties and docking simulation. Based on the QSAR descriptors, the solubility and the hydrophilicity of studied fullerene derivatives increased with increasing the number of oxygen atoms + HMC groups in the compound. While docking calculations indicate that, the compound with two oxygen atoms + HMC groups could interact and binds with HIV-1 protease active site. This is could be attributed to the active site residues of HIV-1 protease are hydrophobic except the two aspartic acids. So that, the increase in the hydrophilicity and polarity of the compound is preventing and/or decreasing the hydrophobic interaction between the compound and HIV-1 protease active site.

  3. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    PubMed

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-04

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties.

  4. Stem Cell Transplant Patients and Fungal Infections

    MedlinePlus

    ... Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch Stem Cell Transplant Patients and Fungal Infections Recommend on Facebook ... Mold . Top of Page Preventing fungal infections in stem cell transplant patients Fungi are difficult to avoid because ...

  5. Subtilases: the superfamily of subtilisin-like serine proteases.

    PubMed Central

    Siezen, R. J.; Leunissen, J. A.

    1997-01-01

    Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling. PMID:9070434

  6. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  7. Bacterial proteases, untapped antimicrobial drug targets.

    PubMed

    Culp, Elizabeth; Wright, Gerard D

    2017-04-01

    Bacterial proteases are an extensive collection of enzymes that have vital roles in cell viability, stress response and pathogenicity. Although their perturbation clearly offers the potential for antimicrobial drug development, both as traditional antibiotics and anti-virulence drugs, they are not yet the target of any clinically used therapeutics. Here we describe the potential for and recent progress in the development of compounds targeting bacterial proteases with a focus on AAA+ family proteolytic complexes and signal peptidases (SPs). Caseinolytic protease (ClpP) belongs to the AAA+ family of proteases, a group of multimeric barrel-shaped complexes whose activity is tightly regulated by associated AAA+ ATPases. The opportunity for chemical perturbation of these complexes is demonstrated by compounds targeting ClpP for inhibition, activation or perturbation of its associated ATPase. Meanwhile, SPs are also a proven antibiotic target. Responsible for the cleavage of targeting peptides during protein secretion, both type I and type II SPs have been successfully targeted by chemical inhibitors. As the threat of pan-antibiotic resistance continues to grow, these and other bacterial proteases offer an arsenal of novel antibiotic targets ripe for development.

  8. Lysosomal protease expression in mature enamel.

    PubMed

    Tye, Coralee E; Lorenz, Rachel L; Bartlett, John D

    2009-01-01

    The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

  9. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  10. Protective immune responses to fungal infections.

    PubMed

    Rivera, A

    2014-09-01

    The incidence of fungal infections has been on the rise over several decades. Fungal infections threaten animals, plants and humans alike and are thus of significant concern to scientists across disciplines. Over the last decade, significant advances on fungal immunology have lead to a better understanding of important mechanisms of host protection against fungi. In this article, I review recent advances of relevant mechanisms of immune-mediated protection to fungal infections.

  11. Fungal diversity in major oil-shale mines in China.

    PubMed

    Jiang, Shaoyan; Wang, Wenxing; Xue, Xiangxin; Cao, Chengyou; Zhang, Ying

    2016-03-01

    As an insufficiently utilized energy resource, oil shale is conducive to the formation of characteristic microbial communities due to its special geological origins. However, little is known about fungal diversity in oil shale. Polymerase chain reaction cloning was used to construct the fungal ribosomal deoxyribonucleic acid internal transcribed spacer (rDNA ITS) clone libraries of Huadian Mine in Jilin Province, Maoming Mine in Guangdong Province, and Fushun Mine in Liaoning Province. Pure culture and molecular identification were applied for the isolation of cultivable fungi in fresh oil shale of each mine. Results of clone libraries indicated that each mine had over 50% Ascomycota (58.4%-98.9%) and 1.1%-13.5% unidentified fungi. Fushun Mine and Huadian Mine had 5.9% and 28.1% Basidiomycota, respectively. Huadian Mine showed the highest fungal diversity, followed by Fushun Mine and Maoming Mine. Jaccard indexes showed that the similarities between any two of three fungal communities at the genus level were very low, indicating that fungi in each mine developed independently during the long geological adaptation and formed a community composition fitting the environment. In the fresh oil-shale samples of the three mines, cultivable fungal phyla were consistent with the results of clone libraries. Fifteen genera and several unidentified fungi were identified as Ascomycota and Basidiomycota using pure culture. Penicillium was the only genus found in all three mines. These findings contributed to gaining a clear understanding of current fungal resources in major oil-shale mines in China and provided useful information for relevant studies on isolation of indigenous fungi carrying functional genes from oil shale.

  12. Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn.

    PubMed

    Ferraretto, L F; Fredin, S M; Shaver, R D

    2015-10-01

    Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated

  13. Fungal endophyte diversity in Sarracenia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal endophytes were isolated from four species of the carnivorous pitcher plant genus Sarracenia: S. minor, S. oreophila, S. purpurea, and S. psittacina. Twelve taxa of fungi, eight within the Ascomycota and four within the Basidiomycota, were identified based on PCR amplification and sequencing ...

  14. Fungal farming in a snail

    PubMed Central

    Silliman, Brian R.; Newell, Steven Y.

    2003-01-01

    Mutualisms between fungi and fungus-growing animals are model systems for studying coevolution and complex interactions between species. Fungal growing behavior has enabled cultivating animals to rise to major ecological importance, but evolution of farming symbioses is thought to be restricted to three terrestrial insect lineages. Surveys along 2,000 km of North America's Atlantic coast documented that the marine snail Littoraria irrorata grazes fungus-infected wounds on live marsh grass throughout its range. Field experiments demonstrate a facultative, farming mutualism between Littoraria and intertidal fungi. Snails graze live grass primarily not to feed but to prepare substrate for fungal growth and consume invasive fungi. Fungal removal experiments show that snails and fungi act synergistically to suppress marsh grass production. These results provide a case of fungus farming in the marine environment and outside the class Insecta and reveal a previously undemonstrated ecological mechanism (i.e., facilitation of fungal invasion) by which grazers can exert top-down control of marine plant production. PMID:14657360

  15. DEMONSTRATION BULLETIN: FUNGAL TREATMENT BULLETIN

    EPA Science Inventory

    Fungal treatment technology uses white rot fungi (lignin degrading fungi) to treat organic contaminated soils in situ. Organic materials inoculated with the fungi are mechanically mixed into the contaminated soil. Using enzymes normally produced for wood degradation as well as ot...

  16. Gene identification and molecular characterization of solvent stable protease from a moderately haloalkaliphilic bacterium, Geomicrobium sp. EMB2.

    PubMed

    Karan, Ram; Singh, Raj Kumar Mohan; Kapoor, Sanjay; Khare, S K

    2011-02-01

    Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively.

  17. Different Culture Metabolites of the Red Sea Fungus Fusarium equiseti Optimize the Inhibition of Hepatitis C Virus NS3/4A Protease (HCV PR)

    PubMed Central

    Hawas, Usama W.; Al-Farawati, Radwan; Abou El-Kassem, Lamia T.; Turki, Adnan J.

    2016-01-01

    The endophytic fungus Fusarium equiseti was isolated from the brown alga Padina pavonica, collected from the Red Sea. The fungus was identified by its morphology and 18S rDNA. Cultivation of this fungal strain in biomalt-peptone medium led to isolation of 12 known metabolites of diketopeprazines and anthraquinones. The organic extract and isolated compounds were screened for their inhibition of hepatitis C virus NS3/4A protease (HCV PR). As a result, the fungal metabolites showed inhibition of HCV protease (IC50 from 19 to 77 μM), and the fungus was subjected to culture on Czapek’s (Cz) media, with a yield of nine metabolites with potent HCV protease inhibition ranging from IC50 10 to 37 μM. The Cz culture extract exhibited high-level inhibition of HCV protease (IC50 27.6 μg/mL) compared to the biomalt culture extract (IC50 56 μg/mL), and the most potent HCV PR isolated compound (Griseoxanthone C, IC50 19.8 μM) from the bio-malt culture extract showed less of an inhibitory effect compared to isolated ω-hydroxyemodin (IC50 10.7 μM) from the optimized Cz culture extract. Both HCV PR active inhibitors ω-hydroxyemodin and griseoxanthone C were considered as the lowest selective safe constituents against Trypsin inhibitory effect with IC50 48.5 and 51.3 μM, respectively. PMID:27775589

  18. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  19. Cleavage entropy as quantitative measure of protease specificity.

    PubMed

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  20. Insect response to plant defensive protease inhibitors.

    PubMed

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

    Plant protease inhibitors (PIs) are natural plant defense proteins that inhibit proteases of invading insect herbivores. However, their anti-insect efficacy is determined not only by their potency toward a vulnerable insect system but also by the response of the insect to such a challenge. Through the long history of coevolution with their host plants, insects have developed sophisticated mechanisms to circumvent antinutritional effects of dietary challenges. Their response takes the form of changes in gene expression and the protein repertoire in cells lining the alimentary tract, the first line of defense. Research in insect digestive proteases has revealed the crucial roles they play in insect adaptation to plant PIs and has brought about a new appreciation of how phytophagous insects employ this group of molecules in both protein digestion and counterdefense. This review provides researchers in related fields an up-to-date summary of recent advances.

  1. Gene targets for fungal and mycotoxin control.

    PubMed

    Kim, J H; Campbell, B C; Molyneux, R; Mahoney, N; Chan, K L; Yu, J; Wilkinson, J; Cary, J; Bhatnagar, D; Cleveland, T E

    2006-03-01

    It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.

  2. Subseafloor basalts as fungal habitats

    NASA Astrophysics Data System (ADS)

    Ivarsson, M.

    2012-09-01

    The oceanic crust is believed to host the largest potential habitat for microbial life on Earth, yet, still we lack substantial information about the abundance, diversity, and consequence of its biosphere. The last two decades have involved major research accomplishments within this field and a change in view of the ocean crust and its potential to harbour life. Here fossilised fungal colonies in subseafloor basalts are reported from three different seamounts in the Pacific Ocean. The fungal colonies consist of various characteristic structures interpreted as fungal hyphae, fruit bodies and spores. The fungal hyphae are well preserved with morphological characteristics such as hyphal walls, septa, thallic conidiogenesis, and hyphal tips with hyphal vesicles within. The fruit bodies consist of large (∼50-200 µm in diameter) body-like structures with a defined outer membrane and an interior filled with calcite. The fruit bodies have at some stage been emptied of their contents of spores and filled by carbonate-forming fluids. A few fruit bodies not filled by calcite and with spores still within support this interpretation. Spore-like structures (ranging from a few µm to ∼20 µm in diameter) are also observed outside of the fruit bodies and in some cases concentrated to openings in the membrane of the fruit bodies. The hyphae, fruit bodies and spores are all closely associated with a crust lining the vein walls that probably represent a mineralized biofilm. The results support a fungal presence in deep subseafloor basalts and indicate that such habitats were vital between ∼81 and 48 Ma.

  3. Serious fungal infections in Thailand.

    PubMed

    Chayakulkeeree, M; Denning, D W

    2017-02-04

    The burden of serious fungal infection in Thailand is increasing but data regarding its incidence and prevalence are lacking. In this study we aimed to estimate the burden of serious fungal diseases in Thailand based on the size of the populations at risk and available epidemiological databases. Data derived from The Bureau of Epidemiology, Department of Disease Control, Thai Ministry of Public Health, World Health Organisation, international and local reports, and some unreported data were used. When no data existed, risk populations were used to estimate frequencies of fungal infections, using previously described methodology by LIFE. Recurrent vulvovaginal candidiasis (>4 episodes per year) is estimated to occur in 3,310 per 100,000 population. Using a previously described rate that 14/10,000 admissions are with fungaemia and 94% of those are Candida, we estimated 8,650 patients with candidaemia. The prevalence of chronic pulmonary aspergillosis is relatively high with a total of 19,044, approximately half subsequent to pulmonary tuberculosis. Invasive aspergillosis is estimated to affect 941 patients following leukaemia therapy, transplantations, and chronic obstructive pulmonary disease, approximately 1.4/100,000. In addition, allergic bronchopulmonary aspergillosis and severe asthma with fungal sensitisation were estimated at approximately 58.4/100,000 and 77/100,000, respectively. Given approximately 8,134 new cases of AIDS annually, cryptococcal meningitis, Pneumocystis pneumonia, and Talaromyces marneffei infection are estimated at 1.9/100,000, 2.6/100,000, and 0.3/100,000, respectively. The present study indicates that about 1.93% (1,254,562) of the population is affected by serious fungal infections. Owing to the lack of data, reports, and statistics, the number of patients with mycoses in Thailand can only be estimated.

  4. Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

    PubMed Central

    Hu, Xin; Compton, Jaimee R.; Leary, Dagmar H.; Olson, Mark A.; Lee, Michael S.; Cheung, Jonah; Ye, Wenjuan; Ferrer, Mark; Southall, Noel; Jadhav, Ajit; Glass, Pamela J.; Marugan, Juan; Legler, Patricia M.

    2017-01-01

    The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-L-methionine dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, 475NVCWAK480, which differs from papain’s (CGS25CWAFS), and the enzyme lacks a transition state (TS) stabilizing residue homologous to Q19 in papain. To understand the roles of conserved residues in catalysis we determined the structure of the free enzyme, and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme, each conformer may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold reductions in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle Eastern Respiratory virus (MERS). Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat

  5. Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

    PubMed Central

    Yanuar, Arry; Suhartanto, Heru; Mun׳im, Abdul; Anugraha, Bram Hik; Syahdi, Rezi Riadhi

    2014-01-01

    HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside. PMID:24616554

  6. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

  7. Seminal and colostral protease inhibitors on leukocytes.

    PubMed

    Veselský, L; Cechová, D; Hruban, V; Klaudy, J

    1982-01-01

    For detection of protease inhibitors from cow colostrum (CTI) and bull seminal plasma (BUSI I and BUSI II) on the surface of leukocytes, immunological methods were used. An agglutination and an immunofluorescence test demonstrated components on the surface of bovine, porcine and ovine granulocytes and lymphocytes which were immunologically identical with the protease inhibitors isolated from cow colostrum and bull seminal plasma. When antisera against (CTI, BUSI and BUSI II were absorbed by bovine and porcine liver, kidney and spleen homogenate or by bovine and porcine granulocytes or lymphocytes, the immunological tests were negative.

  8. Fungal induced corrosion of wire rope exposed in humid atmospheric conditions

    SciTech Connect

    Little, B.; Ray, R.; Hart, K.; Wagner, P.

    1995-03-01

    Localized corrosion of carbon steel wire rope stored in a humid environment on wooden spools was caused by organic acid and carbon dioxide production by fungi growing directly on the wood. Fungal growth was observed on the interiors of wooden spools and corrosion was most severe on the wrap of wire in direct contact with the wood. Laboratory experiments were designed to demonstrate a causal relationship between storage conditions, fungal growth, and localized corrosion.

  9. Detection of protease and protease activity using a single nanoscrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  10. The Chemical Basis of Fungal Bioluminescence.

    PubMed

    Purtov, Konstantin V; Petushkov, Valentin N; Baranov, Mikhail S; Mineev, Konstantin S; Rodionova, Natalja S; Kaskova, Zinaida M; Tsarkova, Aleksandra S; Petunin, Alexei I; Bondar, Vladimir S; Rodicheva, Emma K; Medvedeva, Svetlana E; Oba, Yuichi; Oba, Yumiko; Arseniev, Alexander S; Lukyanov, Sergey; Gitelson, Josef I; Yampolsky, Ilia V

    2015-07-06

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  11. Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.

    PubMed

    Benito, María J; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A; Bermúdez, María E; Córdoba, Juan J

    2002-07-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

  12. Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

    PubMed Central

    Benito, María J.; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A.; Bermúdez, María E.; Córdoba, Juan J.

    2002-01-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products. PMID:12089038

  13. Untargeted metabolic profiling of Vitis vinifera during fungal degradation.

    PubMed

    Karpe, Avinash V; Beale, David J; Morrison, Paul D; Harding, Ian H; Palombo, Enzo A

    2015-05-01

    This paper illustrates the application of an untargeted metabolic profiling analysis of winery-derived biomass degraded using four filamentous fungi (Trichoderma harzianum, Aspergillus niger, Penicillium chrysogenum and P. citrinum) and a yeast (Saccharomyces cerevisiae). Analysis of the metabolome resulted in the identification of 233 significant peak features [P < 0.05; fold change (FC) > 2 and signal-to-noise ratio >50] using gas chromatography-mass spectrometry followed by statistical chemometric analysis. Furthermore, A. niger and P. chrysogenum produced higher biomass degradation due to considerable β-glucosidase and xylanase activities. The major metabolites generated during fungal degradation which differentiated the metabolic profiles of fungi included sugars, sugar acids, organic acids and fatty acids. Although, P. chrysogenum could degrade hemicelluloses due to its high β-glucosidase and xylanase activities, it could not utilize the resultant pentoses, which A. niger and P. citrinum could do efficiently, thus indicating a need of mixed fungal culture to improve the biomass degradation. Saccharomyces cerevisiae, a non-cellulose degrader, exhibited sugar accumulation during the fermentation. Penicillium chrysogenum was observed to degrade about 2% lignin, a property not observed in other fungi. This study emphasized the differential fungal metabolic behavior and demonstrated the potential of metabolomics in optimizing degradation or manipulating pathways to increase yields of products of interest.

  14. Sphingosine-induced apoptosis is dependent on lysosomal proteases.

    PubMed Central

    Kågedal, K; Zhao, M; Svensson, I; Brunk, U T

    2001-01-01

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes. PMID:11583579

  15. Recent patents on microbial proteases for the dairy industry.

    PubMed

    Feijoo-Siota, Lucía; Blasco, Lucía; Rodríguez-Rama, José Luis; Barros-Velázquez, Jorge; Miguel, Trinidad de; Sánchez-Pérez, Angeles; Villa, Tomás G

    2014-01-01

    This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.

  16. Extracellular trypsin-like proteases produced by Cordyceps militaris.

    PubMed

    Hattori, Maki; Isomura, Shigeki; Yokoyama, Eiji; Ujita, Minoru; Hara, Akira

    2005-12-01

    A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.

  17. Sparse Representation for Prediction of HIV-1 Protease Drug Resistance.

    PubMed

    Yu, Xiaxia; Weber, Irene T; Harrison, Robert W

    2013-01-01

    HIV rapidly evolves drug resistance in response to antiviral drugs used in AIDS therapy. Estimating the specific resistance of a given strain of HIV to individual drugs from sequence data has important benefits for both the therapy of individual patients and the development of novel drugs. We have developed an accurate classification method based on the sparse representation theory, and demonstrate that this method is highly effective with HIV-1 protease. The protease structure is represented using our newly proposed encoding method based on Delaunay triangulation, and combined with the mutated amino acid sequences of known drug-resistant strains to train a machine-learning algorithm both for classification and regression of drug-resistant mutations. An overall cross-validated classification accuracy of 97% is obtained when trained on a publically available data base of approximately 1.5×10(4) known sequences (Stanford HIV database http://hivdb.stanford.edu/cgi-bin/GenoPhenoDS.cgi). Resistance to four FDA approved drugs is computed and comparisons with other algorithms demonstrate that our method shows significant improvements in classification accuracy.

  18. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

    PubMed

    Prasad, Rajesh; Atul; Soni, Awakash; Puri, Sunil Kumar; Sijwali, Puran Singh

    2012-01-01

    Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries

  19. Fungal exopolysaccharide: production, composition and applications.

    PubMed

    Mahapatra, Subhadip; Banerjee, Debdulal

    2013-01-01

    Fungal exopolysaccharides (EPSs) have been recognized as high value biomacromolecules for the last two decades. These products, including pullulan, scleroglucan, and botryosphaeran, have several applications in industries, pharmaceuticals, medicine, foods etc. Although fungal EPSs are highly relevant, to date information concerning fungal biosynthesis is scarce and an extensive search for new fugal species that can produce novel EPSs is still needed. In most cases, the molecular weight variations and sugar compositions of fungal EPSs are dependent to culture medium composition and different physical conditions provided during fermentation. An inclusive and illustrative review on fungal EPS is presented here. The general outline of the present work includes fungal EPS production, their compositions and applications. An emphasis is also given to listing out different fungal strains that can produce EPSs.

  20. Fungal Exopolysaccharide: Production, Composition and Applications

    PubMed Central

    Mahapatra, Subhadip; Banerjee, Debdulal

    2013-01-01

    Fungal exopolysaccharides (EPSs) have been recognized as high value biomacromolecules for the last two decades. These products, including pullulan, scleroglucan, and botryosphaeran, have several applications in industries, pharmaceuticals, medicine, foods etc. Although fungal EPSs are highly relevant, to date information concerning fungal biosynthesis is scarce and an extensive search for new fugal species that can produce novel EPSs is still needed. In most cases, the molecular weight variations and sugar compositions of fungal EPSs are dependent to culture medium composition and different physical conditions provided during fermentation. An inclusive and illustrative review on fungal EPS is presented here. The general outline of the present work includes fungal EPS production, their compositions and applications. An emphasis is also given to listing out different fungal strains that can produce EPSs. PMID:24826070

  1. Routes of phlogopite weathering by three fungal strains.

    PubMed

    Pinzari, Flavia; Cuadros, Javier; Napoli, Rosario; Canfora, Loredana; Baussà Bardají, David

    2016-12-01

    Fungi dissolve soil minerals by acidification and mechanical disruption. Dissolution may occur at the microscale (contact between fungus and mineral) and medium scale (entire mineral grains). Mineral weathering by fungi and other microorganisms is thought to be of significant global contribution, perhaps producing specific weathering signatures. We report fungal dissolution of phlogopite mica in experiments with three fungal strains (Alternaria tenuissima, Cladosporium cladosporioides, Stilbella sp.) on solid medium for 30 days at 21 °C and 96-100% relative humidity. The study used variable-pressure SEM-EDS equipped with charge contrast imaging. Statistical analysis of the results discriminated between the weathering activities of the three fungal species, which increased from Stilbella sp. to C. cladosporioides to A. tenuissima, in agreement with the respective decreasing pH in the media (6.4, 5.8, 5.2 ± 0.03). Phlogopite weathering features were irregular and variable, apparently not caused by direct contact with fungal hyphae. EDS values indicated two or more dissolution mechanisms, one of them suggesting cation rearrangement in the mica towards formation of Al-rich smectite. Intimate fungus-mineral interaction was observed, and the lack of observable dissolution traces from such contact interaction is interpreted as the result of effacing by the more intense acid leaching operating at larger scale.

  2. The influence of fig proteases on the inhibition of angiotensin I-converting and GABA formation in meat.

    PubMed

    Li, Jinyue; Izumimoto, Masatoshi; Yonehara, Makiko; Hirotsu, Seiya; Kuriki, Takayoshi; Naito, Ichiro; Yamada, Hidenori

    2009-12-01

    The purposes of this research were to use fig protease for texture tenderizing, and to inhibit angiotensin I-converting enzyme (ACE) action and gamma-aminobutyric acid (GABA) formation of meat. Liberated peptides by the enzymatic action of fig protease in processing meat and free amino acids were determined and ACE inhibitory activity was assayed. Meat treated with fig protease became tender as indicated by shear force value (SFV) which was half of those of non-fig treated meat during storage even at 5 degrees C. Liberated peptides, free amino acids and GABA increased while extremely low levels of Glu were detected after storage. The optimal temperature of fig protease against meat was 80 degrees C. However, the activity of fig protease decreased after pre-heating more than 40 degrees C. High ACE inhibitory activity of a mixture of fig and meat was found around 80 degrees C, and the value corresponded to the amount of liberated peptide. A lot of liberated peptides were found at 60-80 degrees C and pasterization of meat product becomes convenient to produce peptides. Production of ACE inhibitory peptides and GABA can be expected as the healthy functional meat product such as antihypertensive activity and improve brain function.

  3. Effects of an exogenous protease on the fermentation and nutritive value of corn silage harvested at different dry matter contents and ensiled for various lengths of time.

    PubMed

    Windle, M C; Walker, N; Kung, L

    2014-05-01

    The objective of this experiment was to evaluate the effects of adding an experimental protease to corn plants harvested at different maturities on silage fermentation and in vitro ruminal starch digestibility (IVSD). Corn plants were harvested at maturities resulting in plants with 31 or 40% dry matter (DM). Plants were chopped, kernel processed, and treated with (1) only a 0.1 M phosphate buffer (pH 5.5, 5% vol/wt of fresh forage), (2) buffer with protease to obtain a final concentration of 20mg of protease/kg of wet forage, and (3) buffer with protease to obtain a final concentration of 2,000 mg of protease/kg of wet forage. Treated forages (about 500 g) were ensiled in nylon-polyethylene pouches and stored between 21 and 23°C for 0, 45, 90, and 150 d. Data were analyzed as a 2 × 3 × 4 factorial arrangement of treatments, with the main effects of harvest DM, dose of protease, days of ensiling, and their interactions. The treatment with the highest dose of protease resulted in more robust fermentations across harvest DM with higher concentrations of lactic and acetic acids compared with untreated silage. Concentrations of soluble protein (% of crude protein) increased with time of ensiling, regardless of DM content at harvest. However, averaged over both harvest DM contents, it increased by 37% for silages treated with the high dose of protease compared with an average 11% increase for untreated silages and silage treated with the low dose of protease, between d 0 and 45. Averaged over both harvest DM contents, the concentration of soluble protein peaked in silages treated with the high dose of protease after 45 d of ensiling, whereas it peaked at d 90 in untreated silages and silage treated with the low dose of protease. Similar changes occurred in the concentration of NH3-N due to length of ensiling and treatment with protease. In fresh forages, the concentration of starch for early- and late-harvested forages was similar, but IVSD was lower in the latter

  4. Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.

    PubMed

    Hamilton, John M U; Simpson, David J; Hyman, Stefan C; Ndimba, Bongani K; Slabas, Antoni R

    2003-02-15

    A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

  5. Molecular mechanics calculations on Rous sarcoma virus protease with peptide substrates.

    PubMed Central

    Weber, I. T.; Harrison, R. W.

    1997-01-01

    Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and P1'. were built and compared with kinetic measurements. The unsubstituted peptide substrate. Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions. PMID:9385639

  6. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury.

    PubMed

    Murohara, T; Guo, J P; Lefer, A M

    1995-09-01

    Polymorphonuclear neutrophils (PMN) play an important role in myocardial ischemia/reperfusion (MI/R) injury; however, the role of neutrophilic proteases is less understood. The effects of a novel serine protease inhibitor (serpin), LEX032, were investigated in a murine model of MI (20 min) and R (24 hr) injury in vivo. LEX032 is a recombinant human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of alpha-1 protease inhibitor. LEX032 has the ability to inhibit both neutrophil elastase and cathepsin G, two major neutral serine proteases in neutrophils, as well as superoxide generation. LEX032 (25 or 50 mg/kg) administered i.v. 1 min before reperfusion significantly attenuated myocardial necrotic injury evaluated by cardiac creatine kinase loss compared to MI/R rats receiving only vehicle (P < .001). Moreover, cardiac myeloperoxidase activity, an index of PMN accumulation, in the ischemic myocardium was significantly attenuated by LEX032 as compared with rats receiving vehicle (P < .001). LEX032 also moderately attenuated leukotriene B4-stimulated PMN adherence to rat superior mesenteric artery endothelium and markedly diminished superoxide radical release from LTB4-stimulated PMN in vitro. In a glycogen-induced rat peritonitis model, LEX032 (50 mg/kg) significantly attenuated PMN trans