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Sample records for lytic enzymes sleb

  1. Phage lytic enzymes: a history.

    Science.gov (United States)

    Trudil, David

    2015-02-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  2. [Potentialization of antibiotics by lytic enzymes].

    Science.gov (United States)

    Brisou, J; Babin, P; Babin, R

    1975-01-01

    Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.

  3. MUREIN-METABOLIZING ENZYMES FROM ESCHERICHIA-COLI - EXISTENCE OF A 2ND LYTIC TRANSGLYCOSYLASE

    NARCIS (Netherlands)

    ENGEL, H; SMINK, AJ; VANWIJNGAARDEN, L; KECK, W

    1992-01-01

    In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which

  4. Lytic Polysaccharide Monooxygenases - Studies of Fungal Secretomes and Enzyme Properties

    DEFF Research Database (Denmark)

    Nekiunaite, Laura

    degradation, were also identified upstream the LPMO genes, providing evidence for a co-regulatory mechanism of LPMOs and amylolytic hydrolases. The second part of the PhD thesis is focused on understanding the binding properties of LPMOs to starch and starch mimic substrate. It was shown that LPMOs possessing...... to different substrates at the protein level. It could help to design better enzyme cocktails that increase efficiency of biomass degradation. The secretomes of A. nidulans revealed differences in growth and secretion of enzymes, depending on the type and properties of starches. A common characteristic...... conversion as they produce a wide diversity of degrading enzymes. In the first part of this PhD thesis, the secretomes of the well-known fungus Aspergillus nidulans grown on cereal and legume starches were analyzed. Secretomics is a powerful tool to unravel secretion patterns of fungi and their response...

  5. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

  6. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

  7. Bacteriophage and lytic enzymes - can they help us in the war with antibiotic resistant bacteria

    International Nuclear Information System (INIS)

    Trudil, D.; Rainina, E.

    2009-01-01

    Drug-resistant pathogens are a growing menace to all people, regardless of age, or socioeconomic background. They endanger people industrial societies like the United States, as well as in less developed nations and are even causing problems in military field hospitals. From Streptococcus pneumoniae to Staphylococcus, C. difficile, and multidrug-resistant TB, the list is growing. The threat of engineered microorganisms further complicates the interaction between man and Mother Nature. Additionally, although antibiotics were specifically designed for treating human health emergencies, their use for raising livestock animals has expanded. In the US, large amounts of antibiotics are routinely mixed into feed in order to promote growth rather than combat disease and as prophylactic treatment to offset unnatural diets and unhealthy living conditions. U.S.-raised animals in the 1950s received 2 million pounds per year of antibiotics in their feed compared to 50 million pounds today-a 2,500-percent increase. A large percentage of these drugs pass into the environment. In fact, prior to 1995, when fluoroquinolones were first approved to treat poultry, very few fluoroquinolone-resistant Campylobacter were found in people with foodborne diseases in the United States. After the approval, however, many more fluoroquinolone-resistant bacteria were found in humans and in poultry from slaughter plants and retail stores. The threat to our food supply becomes a threat to security. What can be done? One approach is to treat bacterial diseases by the use of bacteriophages. Phages are very small viruses that destroy by lysing select bacteria. The idea of using phage as a therapy for infectious bacterial diseases was first proposed by d'Herelle around World War I and over 80 years bacteriophage has been a key tool of healthcare professionals within Eastern Europe. More recently professionals in the USA and Western Europe have isolated and developed specific lytic components which have

  8. MUREIN-METABOLIZING ENZYMES FROM ESCHERICHIA-COLI - SEQUENCE-ANALYSIS AND CONTROLLED OVEREXPRESSION OF THE SLT GENE, WHICH ENCODES THE SOLUBLE LYTIC TRANSGLYCOSYLASE

    NARCIS (Netherlands)

    ENGEL, H; KAZEMIER, B; KECK, W

    The complete nucleotide sequence of the slt gene encoding the soluble lytic transglycosylase (Slt; EC 3.2.1.-) from Escherichia coli has been determined. The largest open reading frame identified on a 2.5-kb PvuII-SalI fragment indicates that the enzyme is translated as a preprotein of either 654 or

  9. Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

    Science.gov (United States)

    Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

    2017-01-01

    Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

  10. Produção, purificação, clonagem e aplicação de enzimas líticas Production, purification, cloning and application of lytic enzymes

    Directory of Open Access Journals (Sweden)

    Luciana Francisco Fleuri

    2005-10-01

    Full Text Available Lytic enzymes such as beta-1,3 glucanases, proteases and chitinases are able to hydrolyse, respectively, beta-1,3 glucans, mannoproteins and chitin, as well as the cell walls of many yeast species. Lytic enzymes are useful in a great variety of applications including the preparation of protoplasts; the extraction of proteins, enzymes, pigments and functional carbohydrates; pre-treatment for the mechanical rupture of cells; degradation of residual yeast cell mass for the preparation of animal feed; analysis of the yeast cell wall structure and composition; study of the yeast cell wall synthesis and the control of pathogenic fungi. This review presents the most important aspects with respect to lytic enzymes, especially their production, purification, cloning and application.

  11. Biocontrol ability of Lysobacter antibioticus HS124 against Phytophthora blight is mediated by the production of 4-hydroxyphenylacetic acid and several lytic enzymes.

    Science.gov (United States)

    Ko, Hyun-Sun; Jin, Rong-De; Krishnan, Hari B; Lee, Sang-Bog; Kim, Kil-Yong

    2009-12-01

    Several rhizobacteria play a vital role in plant protection, plant growth promotion and the improvement of soil health. In this study, we have isolated a strain of Lysobacter antibioticus HS124 from rhizosphere and demonstrate its antifungal activity against various pathogens including Phytophthora capsici, a destructive pathogen of pepper plants. L. antibioticus HS124 produced lytic enzymes such as chitinase, beta-1,3-glucanase, lipase, protease, and an antibiotic compound. This antibiotic compound was purified by diaion HP-20, silica gel, sephadex LH-20 column chromatography and high performance liquid chromatography. The purified compound was identified as 4-hydroxyphenylacetic acid by gas chromatography-electron ionization (GC-EI) and gas chromatography-chemical ionization (GC-CI) mass spectrometry. This antibiotic exhibited destructive activity toward P. capsici hyphae. In vivo experiments utilizing green house grown pepper plants demonstrated the protective effect of L. antibioticus HS124 against P. capsici. The growth of pepper plants treated with L. antibioticus culture was enhanced, resulting in greater protection from fungal disease. Optimum growth and protection was found when cultures were grown in presence of Fe(III). Additionally, the activities of pathogenesis-related proteins such as chitinase and beta-1,3-glucanase decreased in roots, but increased in leaves with time after treatment compared to controls. Our results demonstrate L. antibioticus HS124 as a promising candidate for biocontrol of P. capsici in pepper plants.

  12. Determination of extra and intracellular content from some lytic enzymes related with carnation (Dianthus caryophyllus L. root cell wall

    Directory of Open Access Journals (Sweden)

    Sixta Tulia Martínez Peralta

    2016-11-01

    Full Text Available The presence of some enzymes related to cell wall (polygalacturonase, the pectate lyase, protease and xylanase in carnation (Dianthus caryophyllus L. roots as well as the activity levels were determined. These levels were analyzed in different cellular places: the intercellular fluid that is part of apoplast, the symplast, and the total level (apoplast and symplast in carnation roots. Two methods were tested to extract the intercellular fluid. To obtain the intracellular content (symplast and total extract (apoplast+symplast, three methods were tested, using as extracting solution  i phosphate buffer, ii phosphate buffer + PVPP,  iii before the extraction with phosphate buffer, the carnation roots were washed with acetone.  The results showed the effect of different extracting solutions in the enzymatic activities and in the protein content. A new only one step method is proposed to extract the four enzymes and make the comparative analysis of enzymatic activity.

  13. Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

    Science.gov (United States)

    Badino, Silke F; Christensen, Stefan J; Kari, Jeppe; Windahl, Michael S; Hvidt, Søren; Borch, Kim; Westh, Peter

    2017-08-01

    Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10 3 -10 4 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Enzyme-driven Bacillus spore coat degradation leading to spore killing.

    Science.gov (United States)

    Mundra, Ruchir V; Mehta, Krunal K; Wu, Xia; Paskaleva, Elena E; Kane, Ravi S; Dordick, Jonathan S

    2014-04-01

    The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. © 2013 Wiley Periodicals, Inc.

  15. Screening of physiologically active strain of the filamentous fungi - a producer of a complex of lytic enzymes

    International Nuclear Information System (INIS)

    Kurbatova, E.I.; Sokolova, E.N.; Borshcheva, Yu.A.; Alsivar, S.K.A.; Rimareva, L.V.

    2014-01-01

    Filamentous Aspergillus fungi were studied to obtain a producer of a complex of the enzymes specific to biodegradation of polymers of cellular walls of vegetable and microbic biomass. Strains were selected by the increased biosynthetic ability in relation to the beta-glucanase (BG), chitinase (CT), mannanase (MN), proteases and pectinases. It was estimated during deep cultivation in the environment containing wheat bran. The fullest complex of hydrolytic enzymes (glucanase, MN, CT, protease and a polygalacturonase (PG)), and also the level of enzymatic activities was in the culture liquid obtained as a result of biosynthesis of Aspergillus foetidus 37-4 (S 37-4) strain. For its cultivation the medium containing salts like potassium dihydrogen phosphate, magnesium sulfate and ammonium sulfate in optimum concentration, and also dioses (maltose, sucrose) and polysaccharides (starch, chitin, pectin) was chosen. The greatest zones of hydrolysis are traced during planting S 37-4 in agar medium containing maltose and low methoxyl citrus pectin. As the synthesis inductor of hemicellulase, MN and CT malt sprouts were used, and of PG - not clarified beet bin fibers. Cultivation was carried out on a thermostatically controlled shaker at 30 deg. C for 120 h. Increase of activity of synthesizable enzymes when using low methoxyl citrus pectin as a media part equaled for BG 5-19%, for PG - 25%, when using a maltose for CT - 100%, MN - 29%. To increase biosynthetic ability of S 37-4 as a mutagen 3-staged ultra-violet radiation (wavelength is 265 nanometers) was applied. The obtained 379-K-5 strain surpassed in activity level a parental strain BG - by 84.8%, CT - by 45.0%, MN - by 62.9%, PG - by 89.0%. The following (4th) stage of radiation led to death of the strain. In comparison with a parental S 37-4 the colony of a mutant strain possessed the bigger size and plentiful formation of an air mycelium, ability to sporogenesis was less expressed

  16. Technological application of an extracellular cell lytic enzyme in xanthan gum clarification Aplicação tecnológica de uma enzima celulolítica para clarificação de goma xantana

    Directory of Open Access Journals (Sweden)

    Suresh Shastry

    2005-03-01

    Full Text Available An extracellular cell lytic enzyme from Pseudomonas sp. was active on heat killed cells of Xanthomonas campestris. The lytic activity caused enzymatic digestion of X.campestris xanthan gum. Digestion was effective for highly viscous native xanthan 2.0% (w/v and 2.5% (w/v commercial Sigma xanthan. Scanning electron microscopy and SDS-PAGE observations confirmed the cell lytic action on X.campestris cells.Uma enzima extracelular celulolítica produzida por Pseudomonas sp. foi ativa sobre células de Xanthomonas campestris mortas pelo calor. A atividade lítica causou a digestão enzimática de goma xantana de X. campestris. A digestão foi eficiente tanto para xantana nativa altamante viscosa (2,0% w/v como para xantana comercial Sigma (2,5% w/v. Observações por microscopia eletrônica de varredura demonstraram a ação celulolítica sobre células de X. campestris.

  17. Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by Aspergillus nidulans grown on different starches

    DEFF Research Database (Denmark)

    Nekiunaite, Laura; Arntzen, Magnus Ø.; Svensson, Birte

    2016-01-01

    of Aspergillus nidulans grown on cereal starches from wheat and high-amylose (HA) maize, as well as legume starch from pea for 5 days. Aspergillus nidulans grew efficiently on cereal starches, whereas growth on pea starch was poor. The secretomes at days 3-5 were starch-type dependent as also reflected...... by amylolytic activity measurements. Nearly half of the 312 proteins in the secretomes were carbohydrate-active enzymes (CAZymes), mostly glycoside hydrolases (GHs) and oxidative auxiliary activities (AAs). The abundance of the GH13 α-amylase (AmyB) decreased with time, as opposed to other starch...

  18. Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

    Science.gov (United States)

    Vetchinkina, Elena; Kupryashina, Maria; Gorshkov, Vladimir; Ageeva, Marina; Gogolev, Yuri; Nikitina, Valentina

    2017-04-01

    The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

  19. Proteins YlaJ and YhcN contribute to the efficiency of spore germination in Bacillus subtilis.

    Science.gov (United States)

    Johnson, Christian L; Moir, Anne

    2017-04-01

    The YlaJ and YhcN spore lipoproteins of Bacillus subtilis contain a common domain, and are of unknown function. Homologues of YlaJ or YhcN are widespread in Bacilli and are also encoded in those Clostridia that use cortex lytic enzymes SleB and CwlJ for cortex hydrolysis during germination. In B. subtilis, we report that single and double mutants lacking YlaJ and/or YhcN show a reduced rate of spore germination in L-alanine, with a delay in loss of heat resistance, release of dipicolinic acid and OD fall. If B. subtilis spores lack the cortex lytic enzyme CwlJ, spore cortex degradation and subsequent outgrowth to form colonies is strictly dependent on the other cortex lytic enzyme SleB, allowing a test of SleB function; in a cwlJ mutant background, the combined loss of both ylaJ and yhcN genes resulted in a spore population in which only 20% of spores germinated and outgrew to form colonies, suggesting that SleB activity is compromised. YlaJ and YhcN have a role in germination that is not yet well defined, but these proteins are likely to contribute, directly or indirectly, to early events in germination, including effective SleB function. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  1. Reactivation and Lytic Replication of Kaposi’s Sarcoma-Associated Herpesvirus: An Update

    Science.gov (United States)

    Aneja, Kawalpreet K.; Yuan, Yan

    2017-01-01

    The life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed

  2. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  3. Endoplasmic reticulum stress causes EBV lytic replication.

    Science.gov (United States)

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K; Rowe, David T; Wadowsky, Robert M; Rosendorff, Adam

    2011-11-17

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

  4. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  5. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

    2017-01-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of ...

  6. Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

    Science.gov (United States)

    Talento, A; Nguyen, M; Law, S; Wu, J K; Poe, M; Blake, J T; Patel, M; Wu, T J; Manyak, C L; Silberklang, M

    1992-12-15

    Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

  7. Isolation of lytic bacteriophage against Vibrio harveyi.

    Science.gov (United States)

    Crothers-Stomps, C; Høj, L; Bourne, D G; Hall, M R; Owens, L

    2010-05-01

    The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Water samples from discharge channels and grow-out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage-enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of

  8. Endoplasmic reticulum stress causes EBV lytic replication

    OpenAIRE

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K.; Rowe, David T.; Wadowsky, Robert M.; Rosendorff, Adam

    2011-01-01

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)...

  9. Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

    Science.gov (United States)

    Tsfasman, Irina M; Lapteva, Yulia S; Krasovskaya, Ludmila A; Kudryakova, Irina V; Vasilyeva, Natalia V; Granovsky, Igor E; Stepnaya, Olga A

    2015-01-01

    Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms. © 2015 S. Karger AG, Basel.

  10. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    Science.gov (United States)

    Johansen, Katja S

    2016-02-01

    The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. © 2016 Authors; published by Portland Press Limited.

  11. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty......-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used...... as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes...

  12. AtlA Functions as a Peptidoglycan Lytic Transglycosylase in the Neisseria gonorrhoeae Type IV Secretion System▿

    OpenAIRE

    Kohler, Petra L.; Hamilton, Holly L.; Cloud-Hansen, Karen; Dillard, Joseph P.

    2007-01-01

    Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We chara...

  13. Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1

    NARCIS (Netherlands)

    Frommhagen, Matthias

    2017-01-01

    Current developments aim at the effective enzymatic degradation of plant biomass polysaccharides into fermentable monosaccharides for biofuels and biochemicals. Recently discovered lytic polysaccharide monooxgygenases (LPMOs) boost the hydrolytic breakdown of lignocellulosic biomass, especially

  14. Painful Lytic Lesions of the Foot : A Case Report

    Directory of Open Access Journals (Sweden)

    R Vaishya

    2015-03-01

    Full Text Available The presence of lytic lesions in the bones of foot raises a number of diagnostic possibilities ranging from infection, inflammatory pathology to neoplastic conditions. Although the radiological picture is not pathognomonic of any pathology, clinical history and histopathological examination can help to clinch the diagnosis. We present a case of multiple lytic lesions of the foot and discuss possible differential diagnoses. The patient was diagnosed as a case of madura foot and the lesions responded to surgical debridement and anti-fungal treatment with a good functional outcome. Madura foot is an uncommon, chronic granulomatous fungal or bacterial infection with a predilection in people who walk barefoot. Although known for a specific geographical distribution, madura foot should be kept as a possible diagnosis in patients presenting with lytic lesions of the foot due to population emigration across the world.

  15. Metastatic Breast Cancer or Multiple Myeloma? Camouflage by Lytic Lesions

    Directory of Open Access Journals (Sweden)

    Bruce Hough

    2010-01-01

    Full Text Available We report a case of a female with stage I infiltrating ductal carcinoma who received adjuvant therapy including trastuzumab. One year later she developed lytic lesions and was retreated with trastuzumab that was held after she developed symptomatic heart failure. Lytic lesions were attributed to relapse of breast cancer, and cardiac failure attributed to prior trastuzumab therapy. After complications necessitated multiple hospitalizations, a further workup revealed that the lytic lesions were not metastatic breast cancer but multiple myeloma. Her advanced multiple myeloma was associated with systemic amyloidosis involving gut and heart, which ultimately led to her demise. This report addresses the pitfalls of overlapping symptoms and the question of which patients with suspected metastatic disease should undergo a biopsy.

  16. Cloning and expression analysis of genes encoding lytic endopeptidases L1 and L5 from Lysobacter sp. strain XL1.

    Science.gov (United States)

    Lapteva, Y S; Zolova, O E; Shlyapnikov, M G; Tsfasman, I M; Muranova, T A; Stepnaya, O A; Kulaev, I S; Granovsky, I E

    2012-10-01

    Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

  17. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

    2017-09-08

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Viral infection model with periodic lytic immune response

    International Nuclear Information System (INIS)

    Wang Kaifa; Wang Wendi; Liu Xianning

    2006-01-01

    Dynamical behavior and bifurcation structure of a viral infection model are studied under the assumption that the lytic immune response is periodic in time. The infection-free equilibrium is globally asymptotically stable when the basic reproductive ratio of virus is less than or equal to one. There is a non-constant periodic solution if the basic reproductive ratio of the virus is greater than one. It is found that period doubling bifurcations occur as the amplitude of lytic component is increased. For intermediate birth rates, the period triplication occurs and then period doubling cascades proceed gradually toward chaotic cycles. For large birth rate, the period doubling cascade proceeds gradually toward chaotic cycles without the period triplication, and the inverse period doubling can be observed. These results can be used to explain the oscillation behaviors of virus population, which was observed in chronic HBV or HCV carriers

  19. Bacteriophage enzymes for the prevention and treatment of bacterial infections: Stability and stabilization of the enzyme lysing Streptococcus pyogenes cells

    Energy Technology Data Exchange (ETDEWEB)

    Klyachko, N. L.; Dmitrieva, N. F.; Eshchina, A. S.; Ignatenko, O. V.; Filatova, L. Y.; Rainina, Evguenia I.; Kazarov, A. K.; Levashov, A. V.

    2008-06-01

    Recombinant, phage associated lytic enzyme Ply C capable to lyse streptococci of groups A and C was stabilized in the variety of the micelles containing compositions to improve the stability of the enzyme for further application in medicine. It was shown that, in the micellar polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions ((pH 6.3, room temperature), it completely lost its activity in 2 days

  20. Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation.

    Science.gov (United States)

    Nocadello, Salvatore; Minasov, George; Shuvalova, Ludmilla S; Dubrovska, Ievgeniia; Sabini, Elisabetta; Anderson, Wayne F

    2016-07-15

    Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater.

    Science.gov (United States)

    Eydal, Hallgerd S C; Jägevall, Sara; Hermansson, Malte; Pedersen, Karsten

    2009-10-01

    Viruses were earlier found to be 10-fold more abundant than prokaryotes in deep granitic groundwater at the Aspö Hard Rock Laboratory (HRL). Using a most probable number (MPN) method, 8-30 000 cells of sulphate-reducing bacteria per ml were found in groundwater from seven boreholes at the Aspö HRL. The content of lytic phages infecting the indigenous bacterium Desulfovibrio aespoeensis in Aspö groundwater was analysed using the MPN technique for phages. In four of 10 boreholes, 0.2-80 phages per ml were found at depths of 342-450 m. Isolates of lytic phages were made from five cultures. Using transmission electron microscopy, these were characterized and found to be in the Podoviridae morphology group. The isolated phages were further analysed regarding host range and were found not to infect five other species of Desulfovibrio or 10 Desulfovibrio isolates with up to 99.9% 16S rRNA gene sequence identity to D. aespoeensis. To further analyse phage-host interactions, using a direct count method, growth of the phages and their host was followed in batch cultures, and the viral burst size was calculated to be approximately 170 phages per lytic event, after a latent period of approximately 70 h. When surviving cells from infected D. aespoeensis batch cultures were inoculated into new cultures and reinfected, immunity to the phages was found. The parasite-prey system found implies that viruses are important for microbial ecosystem diversity and activity, and for microbial numbers in deep subsurface groundwater.

  2. Percutaneous aspiration biopsy in cervical spine lytic lesions

    International Nuclear Information System (INIS)

    Tampieri, D.; Weill, A.; Melanson, D.; Ethier, R.

    1991-01-01

    We describe the technique and the results of the percutaneous aspiration biopsy (PAB) in a series of 9 patients presenting with neck pain and different degrees of myelopathy, in whom the cervical spine X-ray demonstrated lytic lesions of unknown origin. PAB is a useful, relatively safe technique, and leads to histological diagnosis between metastatic and inflammatory processes. Furthermore, in inflammatory lesions with negative hemoculture, PAB may help in detecting the micro-organism responsible and therefore allow a better antibiotic treatment. (orig.)

  3. AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system.

    Science.gov (United States)

    Kohler, Petra L; Hamilton, Holly L; Cloud-Hansen, Karen; Dillard, Joseph P

    2007-08-01

    Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

  4. Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

    Science.gov (United States)

    Hammer, John A

    2018-02-19

    Cytotoxic T cells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact. Published by Elsevier Ltd.

  5. Lytic effects of mixed micelles of fatty acids and bile acids

    NARCIS (Netherlands)

    Lapré, J. A.; Termont, D. S.; Groen, A. K.; van der Meer, R.

    1992-01-01

    It has been hypothesized that bile acids and fatty acids promote colon cancer. A proposed mechanism is a lytic effect of these surfactants on colonic epithelium, resulting in a compensatory proliferation of colonic cells. To investigate the first step of this hypothesis, we studied the lytic

  6. Crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltE from Escherichia coli

    International Nuclear Information System (INIS)

    Artola-Recolons, Cecilia; Llarrull, Leticia I.; Lastochkin, Elena; Mobashery, Shahriar; Hermoso, Juan A.

    2010-01-01

    Crystals of the lytic transglycosylase MltE from E. coli were grown using the microbatch method and diffracted to a resolution of 2.1 Å. MltE from Escherichia coli (193 amino acids, 21 380 Da) is a lytic transglycosylase that initiates the first step of cell-wall recycling. This enzyme is responsible for the cleavage of the cell-wall peptidoglycan at the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units. At the end this reaction generates a disaccharide that is internalized and initiates the recycling process. To obtain insights into the biological functions of MltE, crystallization trials were performed and crystals of MltE protein that were suitable for X-ray diffraction analysis were obtained. The MltE protein of E. coli was crystallized using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture consisting of 28% polyethylene glycol 4000, 0.1 M Tris pH 8.4 and 0.2 M magnesium chloride. Further optimization was performed using the microbatch technique. Single crystals were obtained that belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 123.32, b = 183.93, c = 35.29 Å, and diffracted to a resolution of 2.1 Å

  7. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Maia Merabishvili

    Full Text Available Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively, high burst size (125 and 145, respectively, stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

  8. Innate Lymphoid Cells (ILCs) as Mediators of Inflammation, Release of Cytokines and Lytic Molecules.

    Science.gov (United States)

    Elemam, Noha Mousaad; Hannawi, Suad; Maghazachi, Azzam A

    2017-12-10

    Innate lymphoid cells (ILCs) are an emerging group of immune cells that provide the first line of defense against various pathogens as well as contributing to tissue repair and inflammation. ILCs have been classically divided into three subgroups based on their cytokine secretion and transcription factor profiles. ILC nomenclature is analogous to that of T helper cells. Group 1 ILCs composed of natural killer (NK) cells as well as IFN-γ secreting ILC1s. ILC2s have the capability to produce T H 2 cytokines while ILC3s and lymphoid tissue inducer (LTis) are subsets of cells that are able to secrete IL-17 and/or IL-22. A recent subset of ILC known as ILC4 was discovered, and the cells of this subset were designated as NK17/NK1 due to their release of IL-17 and IFN-γ. In this review, we sought to explain the subclasses of ILCs and their roles as mediators of lytic enzymes and inflammation.

  9. Innate Lymphoid Cells (ILCs as Mediators of Inflammation, Release of Cytokines and Lytic Molecules

    Directory of Open Access Journals (Sweden)

    Noha Mousaad Elemam

    2017-12-01

    Full Text Available Innate lymphoid cells (ILCs are an emerging group of immune cells that provide the first line of defense against various pathogens as well as contributing to tissue repair and inflammation. ILCs have been classically divided into three subgroups based on their cytokine secretion and transcription factor profiles. ILC nomenclature is analogous to that of T helper cells. Group 1 ILCs composed of natural killer (NK cells as well as IFN-γ secreting ILC1s. ILC2s have the capability to produce TH2 cytokines while ILC3s and lymphoid tissue inducer (LTis are subsets of cells that are able to secrete IL-17 and/or IL-22. A recent subset of ILC known as ILC4 was discovered, and the cells of this subset were designated as NK17/NK1 due to their release of IL-17 and IFN-γ. In this review, we sought to explain the subclasses of ILCs and their roles as mediators of lytic enzymes and inflammation.

  10. Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation.

    Science.gov (United States)

    Huang, Lu; Yang, Mengtian; Yuan, Yan; Li, Xiaojuan; Kuang, Ersheng

    2017-02-01

    Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

    Science.gov (United States)

    Kosowicz, John G; Lee, Jaeyeun; Peiffer, Brandon; Guo, Zufeng; Chen, Jianmeng; Liao, Gangling; Hayward, S Diane; Liu, Jun O; Ambinder, Richard F

    2017-08-15

    Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors. Copyright © 2017 American Society for Microbiology.

  12. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    International Nuclear Information System (INIS)

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B.

    2007-01-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells

  13. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

    2017-03-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Potential of a lytic bacteriophage to disrupt Acinetobacter baumannii biofilms in vitro.

    Science.gov (United States)

    Liu, Yannan; Mi, Zhiqiang; Niu, Wenkai; An, Xiaoping; Yuan, Xin; Liu, Huiying; Wang, Yong; Feng, Yuzhong; Huang, Yong; Zhang, Xianglilan; Zhang, Zhiyi; Fan, Hang; Peng, Fan; Li, Puyuan; Tong, Yigang; Bai, Changqing

    2016-10-01

    The ability of Acinetobacter baumannii to form biofilms and develop antibiotic resistance makes it difficult to control infections caused by this bacterium. In this study, we explored the potential of a lytic bacteriophage to disrupt A. baumannii biofilms. The potential of the lytic bacteriophage to disrupt A. baumannii biofilms was assessed by performing electron microscopy, live/dead bacterial staining, crystal violet staining and by determining adenosine triphosphate release. The bacteriophage inhibited the formation of and disrupted preformed A. baumannii biofilms. Results of disinfection assay showed that the lytic bacteriophage lysed A. baumannii cells suspended in blood or grown on metal surfaces. These results suggest the potential of the lytic bacteriophage to disrupt A. baumannii biofilms.

  15. Navigating barriers: the challenge of directed secretion at the natural killer cell lytic immunological synapse.

    Science.gov (United States)

    Sanborn, Keri B; Orange, Jordan S

    2010-05-01

    Natural killer (NK) cells have an inherent ability to recognize and destroy a wide array of cells rendered abnormal by stress or disease. NK cells can kill a targeted cell by forming a tight interface-the lytic immunological synapse. This represents a dynamic molecular arrangement that over time progresses through a series of steps to ultimately deliver the contents of specialized organelles known as lytic granules. In order to mediate cytotoxicity, the NK cell faces the challenge of mobilizing the lytic granules, polarizing them to the targeted cell, facilitating their approximation to the NK cell membrane, and releasing their contents. This review is focused upon the final steps in accessing function through the lytic immunological synapse.

  16. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  17. Liposome Disruption Assay to Examine Lytic Properties of Biomolecules.

    Science.gov (United States)

    Jimah, John R; Schlesinger, Paul H; Tolia, Niraj H

    2017-08-05

    Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al. , 2000; Jimah et al. , 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al. , 2017.

  18. Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

    Science.gov (United States)

    Hogan, Chad H.; Oldenburg, Darby G.; Kara, Mehmet

    2018-01-01

    Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host. PMID:29390024

  19. Coordination of KSHV Latent and Lytic Gene Control by CTCF-Cohesin Mediated Chromosome Conformation

    Science.gov (United States)

    Kang, Hyojeung; Wiedmer, Andreas; Yuan, Yan; Robertson, Erle; Lieberman, Paul M.

    2011-01-01

    Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5′ and 3′ end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control. PMID:21876668

  20. Deletion of Lytic Transglycosylases Increases Beta-Lactam Resistance in Shewanella oneidensis

    Science.gov (United States)

    Yin, Jianhua; Sun, Yiyang; Sun, Yijuan; Yu, Zhiliang; Qiu, Juanping; Gao, Haichun

    2018-01-01

    Production of chromosome-encoded β-lactamases confers resistance to β-lactams in many Gram-negative bacteria. Some inducible β-lactamases, especially the class C β-lactamase AmpC in Enterobacteriaceae, share a common regulatory mechanism, the ampR-ampC paradigm. Induction of ampC is intimately linked to peptidoglycan recycling, and the LysR-type transcriptional regulator AmpR plays a central role in the process. However, our previous studies have demonstrated that the expression of class D β-lactamase gene blaA in Shewanella oneidensis is distinct from the established paradigm since an AmpR homolog is absent and major peptidoglycan recycling enzymes play opposite roles in β-lactamase expression. Given that lytic transglycosylases (LTs), a class of peptidoglycan hydrolases cleaving the β-1,4 glycosidic linkage in glycan strands of peptidoglycan, can disturb peptidoglycan recycling, and thus may affect induction of blaA. In this study, we investigated impacts of such enzymes on susceptibility to β-lactams. Deletion of three LTs (SltY, MltB and MltB2) increased β-lactam resistance, while four other LTs (MltD, MltD2, MltF, and Slt2) seemed dispensable to β-lactam resistance. The double LT mutants ΔmltBΔmltB2 and ΔsltYΔmltB2 had β-lactam resistance stronger than any of the single mutants. Deletion of ampG (encoding permease AmpG) and mrcA (encoding penicillin binding protein 1a, PBP1a) from both double LT mutants further increased the resistance to β-lactams. Notably, all increased β-lactam resistance phenotypes were in accordance with enhanced blaA expression. Although significant, the increase in β-lactamase activity after inactivating LTs is much lower than that produced by PBP1a inactivation. Our data implicate that LTs play important roles in blaA expression in S. oneidensis. PMID:29403465

  1. Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria.

    Science.gov (United States)

    Yosef, Ido; Manor, Miriam; Kiro, Ruth; Qimron, Udi

    2015-06-09

    The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

  2. The Lytic SA Phage Demonstrate Bactericidal Activity against Mastitis Causing Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hamza Ameer

    2016-01-01

    Full Text Available Staphylococcus aureus is the major causative agent of mastitis among dairy animals as it causes intramammary gland infection. Due to antibiotic resistance and contamination of antibiotics in the milk of diseased animals; alternative therapeutic agents are required to cure mastitis. Lytic bacteriophages and their gene products can be potential therapeutic agents against bacteria as they are host specific and less harmful than antibiotics. In this study, Staphylococcus aureus were isolated from milk samples of the infected animals and identified biochemically. SA phage was isolated from sewage water showing lytic activity against Staphylococcus aureus isolates. The highest lytic activity of bacteriophages was observed at 37°C and pH 7, and the most suitable storage condition was at 4°C. SA phage efficiently reduced bacterial growth in the bacterial reduction assay. The characterization and bacterial growth reduction activity of the bacteriophages against Staphylococcus aureus signifies their underlying potential of phage therapy against mastitis.

  3. The importance of lytic and nonlytic immune responses in viral infections

    DEFF Research Database (Denmark)

    Wodarz, Dominik; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2002-01-01

    Antiviral immune effector mechanisms can be divided broadly into lytic and nonlytic components. We use mathematical models to investigate the fundamental question of which type of response is required to combat different types of viral infection. According to our model, the relative roles...... of the two types of component depend on the cytopathicity of the virus relative to its rate of replication. If the viral cytopathicity is low relative to the rate of viral replication, the model predicts that a combination of lytic and nonlytic effector mechanisms is likely to be required to resolve...

  4. Phenoloxidase but not lytic activity reflects resistance against Pasteuria ramosa in Daphnia magna.

    Science.gov (United States)

    Pauwels, Kevin; De Meester, Luc; Decaestecker, Ellen; Stoks, Robby

    2011-02-23

    The field of ecological immunology strongly relies on indicators of immunocompetence. Two major indicators in invertebrates, the activity of phenoloxidase (PO) and lytic activity have recently been questioned in studies showing that, across a natural range of baseline levels, these indicators did not predict resistance against a manipulated challenge with natural parasites. We confirmed this finding by showing that baseline levels of PO and lytic activity in the host Daphnia magna were not related to spore load of the parasite Pasteuria ramosa. Yet, PO levels in infected hosts did predict spore load, indicating PO activity can be useful as an indicator of immunocompetence in this model parasite-host system.

  5. Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

    Science.gov (United States)

    Chiaki Hori; Jill Gaskell; Kiyohiko Igarashi; Masahiro Samejima; David Hibbett; Bernard Henrissat; Dan Cullen

    2013-01-01

    To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (...

  6. Pancreatic Enzymes

    Science.gov (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  7. CELLULOSE DEGRADATION BY OXIDATIVE ENZYMES

    Directory of Open Access Journals (Sweden)

    Maria Dimarogona

    2012-09-01

    Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  8. A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases...... that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes...

  9. Effectiveness of lytic bacteriophages in reducing E. coli O157:H7 populations introduced through cross-contamination on fresh cut lettuce

    Science.gov (United States)

    Previous research has shown that lytic bacteriophages (phages) can kill E. coli O157:H7 on produce surfaces. The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield) at 10^8 PFU/m...

  10. Hemoglobin is a co-factor of human trypanosome lytic factor

    DEFF Research Database (Denmark)

    Widener, Justin; Nielsen, Marianne Jensby; Shiflett, April

    2007-01-01

    Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have be...

  11. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Giuseppe Balistreri

    2016-02-01

    Full Text Available Kaposi's sarcoma herpesvirus (KSHV causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

  12. Role of the adenovirus early region 1B tumor antigens in transformation and lytic infection

    NARCIS (Netherlands)

    Bernards, R.A.; Leeuw, M.G.W. de; Houweling, A.; Eb, A.J. van der

    1986-01-01

    We have investigated the contribution of each of the two adenovirus type 5 (Ad5) major early region lb (Elb) proteins in cell transformation and in lytic infection. An Ad5 El plasmid, in which the reading frame for the 19-kDa Elb protein was abolished by a stop codon close to the initiation codon,

  13. Crystal structure and mechanism of the lytic transglycosylase MltA from Escherichia coli

    NARCIS (Netherlands)

    van Straaten, Karin

    2006-01-01

    This thesis describes the determination and analysis of the 3D-structure of the lytic transglycosylase MltA from Escherichia coli by X-ray crystallography. This work aims to further increase our knowledge of the molecular details of the cleaving mechanism and the typical 1,6- anhydromuropeptide

  14. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry

    DEFF Research Database (Denmark)

    Westereng, Bjørge; Arntzen, Magnus Ø.; Wittrup Agger, Jane

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the num...

  15. Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

    Science.gov (United States)

    Solís-Sánchez, Alejandro; Hernández-Chiñas, Ulises; Navarro-Ocaña, Armando; De la Mora, Javier; Xicohtencatl-Cortes, Juan; Eslava-Campos, Carlos

    2016-03-22

    Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. ØVC8 is a lytic phage with specific activity against V. cholerae

  16. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases

    DEFF Research Database (Denmark)

    Yuhong, Huang; Busk, Peter Kamp; Lange, Lene

    2015-01-01

    in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose...

  17. Classification of lipolytic enzymes and their biotechnological applications in the pulping industry

    CSIR Research Space (South Africa)

    Ramnath, L

    2017-03-01

    Full Text Available are very closely related (Lee 2016). Enzymes exhibit the canon- ical�/�-hydrolase fold and contain a typical catalytic triad. High activities at low temperature (less than 15 °C) were believed to originate from conserved sequence motifs of these enzymes... enzymes to a family. However, unique families are being discovered through the use of metagenomics (Fu et al. 2011; Kim et al. 2009; Lee et al. 2006). Table 1 summarizes the different classes of lipo- lytic enzymes currently described. Lipases Lipases (e...

  18. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence....... In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...

  19. Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection.

    Science.gov (United States)

    Chijioke, Obinna; Müller, Anne; Feederle, Regina; Barros, Mario Henrique M; Krieg, Carsten; Emmel, Vanessa; Marcenaro, Emanuela; Leung, Carol S; Antsiferova, Olga; Landtwing, Vanessa; Bossart, Walter; Moretta, Alessandro; Hassan, Rocio; Boyman, Onur; Niedobitek, Gerald; Delecluse, Henri-Jacques; Capaul, Riccarda; Münz, Christian

    2013-12-26

    Primary infection with the human oncogenic Epstein-Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Human Natural Killer Cells Prevent Infectious Mononucleosis Features by Targeting Lytic Epstein-Barr Virus Infection

    Directory of Open Access Journals (Sweden)

    Obinna Chijioke

    2013-12-01

    Full Text Available Primary infection with the human oncogenic Epstein-Barr virus (EBV can result in infectious mononucleosis (IM, a self-limiting disease caused by massive lymphocyte expansion that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies.

  1. Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Yuan Yihui

    2012-12-01

    Full Text Available Abstract Background Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33. Results Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins. Conclusions PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

  2. Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

    OpenAIRE

    Balistreri, Giuseppe; Viiliainen, Johanna; Turunen, Mikko; Diaz, Raquel; Lyly, Lauri; Pekkonen, Pirita; Rantala, Juha; Ojala, Krista; Sarek, Grzegorz; Teesalu, Mari; Denisova, Oxana; Peltonen, Karita; Julkunen, Ilkka; Varjosalo, Markku; Kainov, Denis

    2016-01-01

    Kaposi?s sarcoma herpesvirus (KSHV) causes Kaposi?s sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored...

  3. Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication.

    Science.gov (United States)

    Bilger, Andrea; Plowshay, Julie; Ma, Shidong; Nawandar, Dhananjay; Barlow, Elizabeth A; Romero-Masters, James C; Bristol, Jillian A; Li, Zhe; Tsai, Ming-Han; Delecluse, Henri-Jacques; Kenney, Shannon C

    2017-07-04

    EBV infection causes mononucleosis and is associated with specific subsets of B cell lymphomas. Immunosuppressed patients such as organ transplant recipients are particularly susceptible to EBV-induced lymphoproliferative disease (LPD), which can be fatal. Leflunomide (a drug used to treat rheumatoid arthritis) and its active metabolite teriflunomide (used to treat multiple sclerosis) inhibit de novo pyrimidine synthesis by targeting the cellular dihydroorotate dehydrogenase, thereby decreasing T cell proliferation. Leflunomide also inhibits the replication of cytomegalovirus and BK virus via both "on target" and "off target" mechanisms and is increasingly used to treat these viruses in organ transplant recipients. However, whether leflunomide/teriflunomide block EBV replication or inhibit EBV-mediated B cell transformation is currently unknown. We show that teriflunomide inhibits cellular proliferation, and promotes apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose. In addition, teriflunomide prevents the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model. Furthermore, teriflunomide inhibits lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation, and by blocking lytic viral DNA replication. Leflunomide/teriflunomide might therefore be clinically useful for preventing EBV-induced LPD in patients who have high EBV loads yet require continued immunosuppression.

  4. Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

    Science.gov (United States)

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

    Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?

    Directory of Open Access Journals (Sweden)

    Diana Gutiérrez

    2018-01-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs] have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials.

  6. Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

    Energy Technology Data Exchange (ETDEWEB)

    O; Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.; Meilleur, Flora (ORNL); (NCSU)

    2017-01-19

    Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

  7. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  8. Anti-TNFα therapy for inflammatory bowel diseases is associated with Epstein-Barr virus lytic activation.

    Science.gov (United States)

    Lapsia, Sameer; Koganti, Siva; Spadaro, Salvatore; Rajapakse, Ramona; Chawla, Anupama; Bhaduri-McIntosh, Sumita

    2016-02-01

    Anti-TNFα therapy, known to suppress T-cell immunity, is increasingly gaining popularity for treatment of autoimmune diseases including inflammatory bowel diseases (IBD). T-cell suppression increases the risk of B-cell EBV-lymphoproliferative diseases and lymphomas. Since EBV-lytic activation is essential for development of EBV-lymphomas and there have been reports of EBV-lymphomas in patients treated with anti-TNFα therapy, we investigated if patients treated with anti-TNFα antibodies demonstrate greater EBV-lytic activity in blood. Peripheral blood mononuclear cells from 10 IBD patients solely on anti-TNFα therapy compared to 3 control groups (10 IBD patients not on immunosuppressive therapy, 10 patients with abdominal pain but without IBD, and 10 healthy subjects) were examined for the percentage of T-cells, EBV load and EBV-lytic transcripts. Patients on anti-TNFα therapy had significantly fewer T-cells, greater EBV load, and increased levels of transcripts from EBV-lytic genes of all kinetic classes compared to controls. Furthermore, exposure of EBV-infected B-cell lines to anti-TNFα antibodies resulted in increased levels of BZLF1 mRNA; BZLF1 encodes for ZEBRA, the viral latency-to-lytic cycle switch. Thus, IBD patients treated with anti-TNFα antibodies have greater EBV loads likely due to enhanced EBV-lytic gene expression and anti-TNFα antibodies may be sufficient to activate the EBV lytic cycle. Findings from this pilot study lay the groundwork for additional scientific and clinical investigation into the effects of anti-TNFα therapy on the life cycle of EBV, a ubiquitous oncovirus that causes lymphomas in the setting of immunocompromise. © 2015 Wiley Periodicals, Inc.

  9. Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.; Payne, Christina M.

    2015-05-21

    In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending on enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases

  10. A Cell Culture Model of Latent and Lytic Herpes Simplex Virus Type 1 Infection in Spiral Ganglion.

    Science.gov (United States)

    Liu, Yuehong; Li, Shufeng

    2015-01-01

    Reactivation of latent herpes simplex virus type 1 (HSV-1) in spiral ganglion neurons (SGNs) is supposed to be one of the causes of idiopathic sudden sensorineural hearing loss. This study aims to establish a cell culture model of latent and lytic HSV-1 infection in spiral ganglia. In the presence of acyclovir, primary cultures of SGNs were latently infected with HSV-1 expressing green fluorescent protein. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. TCID50 was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency-associated transcript (LAT) using reverse transcription polymerase chain reaction. There is no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA-treated cultures. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA-treated cultures. Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.

  11. Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?

    Science.gov (United States)

    Gutiérrez, Diana; Fernández, Lucía; Rodríguez, Ana; García, Pilar

    2018-01-23

    Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs]) have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials. Copyright © 2018 Gutiérrez et al.

  12. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  13. Radiation-induced focal cortical necrosis of the femur presenting as a lytic lesion

    Energy Technology Data Exchange (ETDEWEB)

    Ilaslan, Hakan; Schils, Jean [Cleveland Clinic, Musculoskeletal Radiology, Cleveland, OH (United States); Joyce, Michael [Cleveland Clinic, Orthopedic Oncology, Cleveland, OH (United States); Shah, Chirag [Cleveland Clinic, Radiation Oncology, Cleveland, OH (United States); Zhang, Yaxia [Cleveland Clinic, Pathology, Cleveland, OH (United States)

    2017-11-15

    Management of soft tissue sarcomas is often complicated, requiring radiation before and in some cases after limb-sparing surgery. Radiation necrosis is a severe complication after radiation treatment and is typically dose related and involves medullary bone. We report on two cases of hitherto unreported focal circumscribed intra-cortical lytic lesions within the radiation portal, which appeared 19 months and 31 months, respectively, after the conclusion of radiation treatment. Both patients had a history of soft tissue sarcoma treated with radiation (66 Gy) and surgical resection. Biopsy of these lesions showed necrotic bone attributed to radiation. (orig.)

  14. Percutaneous aspiration biopsy in cervical spine lytic lesions. Indications and technique

    Energy Technology Data Exchange (ETDEWEB)

    Tampieri, D; Weill, A; Melanson, D; Ethier, R [Montreal Neurological Inst. and Hospital, PQ (Canada). Dept. of Neuroradiology

    1991-02-01

    We describe the technique and the results of the percutaneous aspiration biopsy (PAB) in a series of 9 patients presenting with neck pain and different degrees of myelopathy, in whom the cervical spine X-ray demonstrated lytic lesions of unknown origin. PAB is a useful, relatively safe technique, and leads to histological diagnosis between metastatic and inflammatory processes. Furthermore, in inflammatory lesions with negative hemoculture, PAB may help in detecting the micro-organism responsible and therefore allow a better antibiotic treatment. (orig.).

  15. Protozoacidal Trojan-Horse: use of a ligand-lytic peptide for selective destruction of symbiotic protozoa within termite guts.

    Science.gov (United States)

    Sethi, Amit; Delatte, Jennifer; Foil, Lane; Husseneder, Claudia

    2014-01-01

    For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a 'Trojan-Horse' that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite's gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.

  16. Epstein-Barr virus (EBV Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

    Directory of Open Access Journals (Sweden)

    Yen-Ju Chen

    Full Text Available Epstein-Barr virus (EBV Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV, to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1 an ideal environment for virus reactivation if EBV or KSHV coexists and (2 a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

  17. Effect of metals on the lytic cycle of the coccolithovirus, EhV86.

    Directory of Open Access Journals (Sweden)

    Martha eGledhill

    2012-04-01

    Full Text Available In this study we show that metals, and in particular copper (Cu, can disrupt the lytic cycle in the Emiliania huxleyi - EhV86 host-virus system. Numbers of virus particles produced per E. huxleyi cell and E. huxleyi lysis rates were reduced by Cu at total metal concentrations over 500 nM in the presence of EDTA (ethylenediaminetetraacetic acid, and 250 nM in the absence of EDTA in acute short term exposure experiments. Zinc (Zn, cadmium (Cd and cobalt (Co were not observed to affect the lysis rate of EhV86 in these experiments. The cellular glutathione (GSH content increased in virus infected cells, but not as a result of metal exposure. In contrast, the cellular content of phytochelatins (PCs increased only in response to metal exposure. The increase in gluthatione content is consistent with increases in the production of reactive oxygen species (ROS on viral infection, while increases in PC content are likely linked to metal homeostasis and indicate that metal toxicity to the host was not affected by viral infection. We propose that Cu prevents lytic production of EhV86 by interfering with virus DNA (deoxyribonucleic acid synthesis through a transcriptional block, which ultimately suppresses the formation of ROS, a biochemical response required for successful virus infection.

  18. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

    Directory of Open Access Journals (Sweden)

    Ina Ersing

    2017-05-01

    Full Text Available Epstein-Barr virus (EBV replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

  19. Characterization and lytic activity of methicillin-resistant Staphylococcus aureus(MRSA phages isolated from NICU

    Directory of Open Access Journals (Sweden)

    Golnar Rahimzadeh

    2016-06-01

    Full Text Available Background Methicillin-resistant Staphylococcus aureus (MRSA is a well-known pathogen that causes serious diseases in humans. As part of the efforts to control this pathogen, an isolated bacteriophage, Siphoviridae, which specifically targets Methicillin-resistant Staphylococcus aureus (MRSA, was characterized. Aims The objective of this study was to characterize of a virulent bacteriophage (Siphoviridae isolated from a NICU bathroom sink. Methods The MRSA strain was isolated from patient blood. The isolated strain was confirmed as MRSA using conventional methods. Phages were isolated from a NICU bathroom sink and activity was lytic as determined by spot test. Titer phage lysate was measured by the Double Layer Agar (DLA technique. The morphology was found with electron microscopy. The single-step growth curve was plotted. Results Electron microscopy showed the phage as a member of the family Siphoviridae, serogroup A and F. The isolated phage was capable of lytic activity against methicillin-resistant Staphylococcus aureus (MRSA strain as shown by spot test. By DLA, the titre of the phages was determined to be 10×108PFU/ml. The single-step growth curve showed that the latent period of the isolated bacteriophage was 30 min and the total number of viable progeny per infected host, burst size, was 2600 PFU/infected host. Conclusion In this study, two phages were isolated and characterized from a NICU bathroom sink, from the Siphoviridae family, which specifically targetsmethicillin-resistant Staphylococcus aureus (MRSA.

  20. PARTIAL CHARACTERIZATION OF A LYTIC METHICILLIN RESISTANT-STAPHYLOCOCCUS AUREUS BACTERIOPHAGE

    Directory of Open Access Journals (Sweden)

    Sulaiman Al-Yousef

    2014-12-01

    Full Text Available A marked increase in the infection incidence caused by methicillin-resistant Staphylococcus aureus (MRSA strains has been noted in medical practice in recent years. This study was conducted to study the biological and characterize of MRSA-phage. Methicillin resistance of Staphylococcus aureus was detected and confirmed by determining of the MIC of oxacillin by the standard agar dilution method. Phage was biologically purified using single plaque technique, then phage characterization were studied using host range, adsorption time, particle morphology and its structural protein. MRSA phage showing lytic nature was purified by repeated plating after picking of single isolated plaques. This phage is active against all 11 isolates either of S. aureus or MRSA tested as hosts. Phage produced clear plaques indicating their lytic nature. This phage was concentrated employing polyethylene glycol (PEG-NaCl precipitation method. Morphologically, MRSA Phage has a hexagonal head having a long non-contractile tail, indicating his icosahedral nature. Adsorption studies showed 100% adsorption of MRSA-Phage after 35 minutes of exposure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE experimentation indicated that the phage particles contain one major structural protein (about 30 Kda.

  1. Bronchogenic adenocarcinoma presenting as a synchronous solitary lytic skull lesion with ischaemic stroke--case report and literature review.

    LENUS (Irish Health Repository)

    O'Connell, David

    2011-01-01

    The authors describe a rare case of metastatic bronchogenic adenocarcinoma in a 55-year-old man presenting with concomittant solitary lytic skull lesion and ischaemic stroke. Metastatic bronchogenic carcinoma is known to present as lytic skull lesions. Primary brain tumours are also known to cause ischaemic brain injury. An underlying stroke risk may be exagerated by cranial tumour surgery. Patients with brain tumours are well known to be predisposed to an increased risk of developing thromboembolic disease. It is unusual to see metastatic bronchogenic adenocarcinoma presenting as ischaemic stroke with a background of concomittant cerebral metastasis. The aetio-pathogenesis of this rare occurrence is discussed with a review of literature.

  2. The use of lytic bacteriophages to reduce E. coli O157:H7 on fresh cut lettuce introduced through cross-contamination

    Science.gov (United States)

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield) at 108 PFU/ml or a control (phosphate buffered saline, PBS) was applied to lettuce by either 1) spraying on to lettuce piec...

  3. Augmentation of failed human vertebrae with critical un-contained lytic defect restores their structural competence under functional loading: An experimental study.

    Science.gov (United States)

    Alkalay, Ron N; von Stechow, Dietrich; Hackney, David B

    2015-07-01

    Lytic spinal lesions reduce vertebral strength and may result in their fracture. Vertebral augmentation is employed clinically to provide mechanical stability and pain relief for vertebrae with lytic lesions. However, little is known about its efficacy in strengthening fractured vertebrae containing lytic metastasis. Eighteen unembalmed human lumbar vertebrae, having simulated uncontained lytic defects and tested to failure in a prior study, were augmented using a transpedicular approach and re-tested to failure using a wedge fracture model. Axial and moment based strength and stiffness parameters were used to quantify the effect of augmentation on the structural response of the failed vertebrae. Effects of cement volume, bone mineral density and vertebral geometry on the change in structural response were investigated. Augmentation increased the failed lytic vertebral strength [compression: 85% (Paugmentation is effective in bolstering the failed lytic vertebrae compressive and axial structural competence, showing strength estimates up to 50-90% of historical values of osteoporotic vertebrae without lytic defects. This modest increase suggests that lytic vertebrae undergo a high degree of structural damage at failure, with strength only partially restored by vertebral augmentation. The positive effect of cement volume is self-limiting due to extravasation. Copyright © 2015. Published by Elsevier Ltd.

  4. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  5. Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

    Directory of Open Access Journals (Sweden)

    Rosalind A. Gilbert

    2017-12-01

    Full Text Available The rumen is known to harbor dense populations of bacteriophages (phages predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

  6. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

    Science.gov (United States)

    Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

    2004-12-24

    The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

  7. Epiphyseal involvement in Erdheim-Chester disease: radiographic and scintigraphic findings in a case with lytic lesions

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz-Hernandez, G.; Tajahuerce-Romera, G.M.; Latorre-Ibanez, M.D.; Lara-Pomares, A. [Servicio de Medicina Nuclear, Hospital Provincial de Castellon (Spain); Vila-Fayos, V. [Servicio de Reumatologia, Hospital Comarcal de Vinaroz (Spain)

    2000-08-01

    We reported a symmetric increase of activity in lower links secondary to Erdheim-Chester disease and demonstrated by bone scans and radiographs. An inusual scintigraphic and radiographic appearance with epiphyseal involvement and lytic lesions is described. Differential diagnosis of bone scan and radiographic findings is discussed. (orig.)

  8. Lytic Infection of Lactococcus lactis by Bacteriophages Tuc2009 and c2 Triggers Alternative Transcriptional Host Responses

    NARCIS (Netherlands)

    Ainsworth, S.; Zomer, A.L.; Mahony, J.; Sinderen, D. van

    2013-01-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed

  9. Needle aspiration biopsy in the diagnosis of lytic bone lesions in histiocytosis X, Ewing's sarcoma and neuroblastoma

    International Nuclear Information System (INIS)

    Thommesen, P.; Frederiksen, P.; Loewhagen, T.; Willems, J.S.

    1978-01-01

    Cytologic smears obtained by needle aspiration biopsy of lytic bone lesions in 15 patients with histiocytosis X, Ewing's sarcoma and neuroblastoma were reviewed. After conventional staining, histiocytosis X could be diagnosed and differentiated from small cell tumours such as Ewing's sarcoma and neuroblastoma. The need for sampling material for cytochemical and ultrastructural analysis of these small cell tumours by needle aspiration is emphasized. (Auth.)

  10. Clinical Manifestations of Kaposi Sarcoma Herpesvirus Lytic Activation: Multicentric Castleman Disease (KSHV-MCD) and the KSHV Inflammatory Cytokine Syndrome.

    Science.gov (United States)

    Polizzotto, Mark N; Uldrick, Thomas S; Hu, Duosha; Yarchoan, Robert

    2012-01-01

    Soon after the discovery of Kaposi sarcoma (KS)-associated herpesvirus (KSHV), it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD) arising in patients infected with human immunodeficiency virus. It has subsequently been recognized that KSHV-MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV-MCD is dominated by systemic inflammatory symptoms including fevers, cachexia, and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV-MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6). Patients with KSHV-MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h) IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV-MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS) has been described. Its clinical manifestations resemble those of KSHV-MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV-MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, homolog of human interleukin-6 and IL-10 comparable to those seen in KSHV-MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms seen in some patients with

  11. Clinical Manifestations of Kaposi Sarcoma Herpesvirus Lytic Activation: Multicentric Castleman Disease (KSHV–MCD) and the KSHV Inflammatory Cytokine Syndrome

    Science.gov (United States)

    Polizzotto, Mark N.; Uldrick, Thomas S.; Hu, Duosha; Yarchoan, Robert

    2012-01-01

    Soon after the discovery of Kaposi sarcoma (KS)-associated herpesvirus (KSHV), it was appreciated that this virus was associated with most cases of multicentric Castleman disease (MCD) arising in patients infected with human immunodeficiency virus. It has subsequently been recognized that KSHV–MCD is a distinct entity from other forms of MCD. Like MCD that is unrelated to KSHV, the clinical presentation of KSHV–MCD is dominated by systemic inflammatory symptoms including fevers, cachexia, and laboratory abnormalities including cytopenias, hypoalbuminemia, hyponatremia, and elevated C-reactive protein. Pathologically KSHV–MCD is characterized by polyclonal, IgM-lambda restricted plasmacytoid cells in the intrafollicular areas of affected lymph nodes. A portion of these cells are infected with KSHV and a sizable subset of these cells express KSHV lytic genes including a viral homolog of interleukin-6 (vIL-6). Patients with KSHV–MCD generally have elevated KSHV viral loads in their peripheral blood. Production of vIL-6 and induction of human (h) IL-6 both contribute to symptoms, perhaps in combination with overproduction of IL-10 and other cytokines. Until recently, the prognosis of patients with KSHV–MCD was poor. Recent therapeutic advances targeting KSHV-infected B cells with the anti-CD20 monoclonal antibody rituximab and utilizing KSHV enzymes to target KSHV-infected cells have substantially improved patient outcomes. Recently another KSHV-associated condition, the KSHV inflammatory cytokine syndrome (KICS) has been described. Its clinical manifestations resemble those of KSHV–MCD but lymphadenopathy is not prominent and the pathologic nodal changes of KSHV–MCD are absent. Patients with KICS exhibit elevated KSHV viral loads and elevation of vIL-6, homolog of human interleukin-6 and IL-10 comparable to those seen in KSHV–MCD; the cellular origin of these is a matter of investigation. KICS may contribute to the inflammatory symptoms seen in some

  12. Chaetocin reactivates the lytic replication of Epstein-Barr virus from latency via reactive oxygen species.

    Science.gov (United States)

    Zhang, Shilun; Yin, Juan; Zhong, Jiang

    2017-01-01

    Oxidative stress, regarded as a negative effect of free radicals in vivo, takes place when organisms suffer from harmful stimuli. Some viruses can induce the release of reactive oxygen species (ROS) in infected cells, which may be closely related with their pathogenicity. In this report, chaetocin, a fungal metabolite reported to have antimicrobial and cytostatic activity, was studied for its effect on the activation of latent Epstein-Barr virus (EBV) in B95-8 cells. We found that chaetocin remarkably up-regulated EBV lytic transcription and DNA replication at a low concentration (50 nmol L -1 ). The activation of latent EBV was accompanied by an increased cellular ROS level. N-acetyl-L-cysteine (NAC), an ROS inhibitor, suppressed chaetocin-induced EBV activation. Chaetocin had little effect on histone H3K9 methylation, while NAC also significantly reduced H3K9 methylation. These results suggested that chaetocin reactivates latent EBV primarily via ROS pathways.

  13. Isolation and life cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria

    DEFF Research Database (Denmark)

    Middelboe, Mathias; Chan, Amy; Bertelsen, Sif Koldborg

    2010-01-01

    Basic knowledge on viruses infecting heterotrophic bacteria and cyanobacteria is key to future progress in understanding the role of viruses in aquatic systems and the influence of virus–host interactions on microbial mortality, biogeochemical cycles, and genetic exchange. Such studies require......, and discusses the applications and limitations of different isolation procedures. Most work on phage isolation has been carried out with aerobic heterotrophic bacteria and cyanobacteria, culturable both on agar plates and in enriched liquid cultures. The procedures presented here are limited to lytic viruses...... infecting such hosts. In addition to the isolation procedures, methods for life cycle characterization (one-step growth experiments) of bacteriophages and cyanophages are described. Finally, limitations and drawbacks of the proposed methods are assessed and discussed...

  14. Delta-9 tetrahydrocannabinol (THC inhibits lytic replication of gamma oncogenic herpesviruses in vitro

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    Friedman Herman

    2004-09-01

    Full Text Available Abstract Background The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC, has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Methods Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV and Epstein-Barr virus (EBV replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS of monkeys, murine gamma herpesvirus 68 (MHV 68, and herpes simplex type 1 (HSV-1 was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Results Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. Conclusions THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC

  15. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

    Directory of Open Access Journals (Sweden)

    Marie Samanovic

    2009-01-01

    Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

  16. Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro.

    Science.gov (United States)

    Medveczky, Maria M; Sherwood, Tracy A; Klein, Thomas W; Friedman, Herman; Medveczky, Peter G

    2004-09-15

    The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development

  17. Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

    Science.gov (United States)

    Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

    2014-01-01

    CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEevasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens. PMID:25144360

  18. Activation and Repression of Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycles by Short- and Medium-Chain Fatty Acids

    Science.gov (United States)

    Gorres, Kelly L.; Daigle, Derek; Mohanram, Sudharshan

    2014-01-01

    ABSTRACT The lytic cycles of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are induced in cell culture by sodium butyrate (NaB), a short-chain fatty acid (SCFA) histone deacetylase (HDAC) inhibitor. Valproic acid (VPA), another SCFA and an HDAC inhibitor, induces the lytic cycle of KSHV but blocks EBV lytic reactivation. To explore the hypothesis that structural differences between NaB and VPA account for their functional effects on the two related viruses, we investigated the capacity of 16 structurally related short- and medium-chain fatty acids to promote or prevent lytic cycle reactivation. SCFAs differentially affected EBV and KSHV reactivation. KSHV was reactivated by all SCFAs that are HDAC inhibitors, including phenylbutyrate. However, several fatty acid HDAC inhibitors, such as isobutyrate and phenylbutyrate, did not reactivate EBV. Reactivation of KSHV lytic transcripts could not be blocked completely by any fatty acid tested. In contrast, several medium-chain fatty acids inhibited lytic activation of EBV. Fatty acids that blocked EBV reactivation were more lipophilic than those that activated EBV. VPA blocked activation of the BZLF1 promoter by NaB but did not block the transcriptional function of ZEBRA. VPA also blocked activation of the DNA damage response that accompanies EBV lytic cycle activation. Properties of SCFAs in addition to their effects on chromatin are likely to explain activation or repression of EBV. We concluded that fatty acids stimulate the two related human gammaherpesviruses to enter the lytic cycle through different pathways. IMPORTANCE Lytic reactivation of EBV and KSHV is needed for persistence of these viruses and plays a role in carcinogenesis. Our direct comparison highlights the mechanistic differences in lytic reactivation between related human oncogenic gammaherpesviruses. Our findings have therapeutic implications, as fatty acids are found in the diet and produced by the human microbiota

  19. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    Science.gov (United States)

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  20. Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

    Science.gov (United States)

    Husseneder, Claudia; Sethi, Amit; Foil, Lane; Delatte, Jennifer

    2010-12-29

    We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic

  1. Structural and molecular dynamics studies of a C1-oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bing [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Kognole, Abhishek A. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Wu, Miao [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Westereng, Bjørge [Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås Norway; Crowley, Michael F. [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Kim, Seonah [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Dimarogona, Maria [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Department of Chemical Engineering, University of Patras, Greece; Payne, Christina M. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Directorate of Engineering, Division of Chemical, Bioengineering, Environmental, and Transport Systems, National Science Foundation, Alexandria VA USA; Sandgren, Mats [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden

    2018-04-24

    Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 A resolution using X-ray crystallography. Unlike the majority of the currently available C1-specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting ..beta..-strands ..beta..2 and ..beta..3 of the ..beta..-sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active-site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1-specific LPMOs, giving a molecular-level picture of active site substrate interactions.

  2. Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Daniela F Angelini

    Full Text Available It has long been known that multiple sclerosis (MS is associated with an increased Epstein-Barr virus (EBV seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A and lytic (BZLF-1, BMLF-1 antigens in relapsing-remitting MS patients (n = 113 and healthy donors (HD (n = 43 and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

  3. Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Poulsen, Jens-Christian Navarro; Tandrup, Tobias

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) have been found to be key components in microbial (bacterial and fungal) degradation of biomass. They are copper metalloenzymes that degrade polysaccharides oxidatively and act in synergy with glycoside hydrolases. Recently crystallographic studies...

  4. Utility of lytic bacteriophage in the treatment of multidrug-resistant Pseudomonas aeruginosa septicemia in mice

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    Vinodkumar C

    2008-07-01

    Full Text Available Drug resistance is the major cause of increase in morbidity and mortality in neonates. One thousand six hundred forty-seven suspected septicemic neonates were subjected for microbiological analysis over a period of 5 years. Forty-two P. aeruginosa were isolated and the antibiogram revealed that 28 P. aeruginosa were resistant to almost all the common drugs used (multidrug-resistant. The emergence of antibiotic-resistant bacterial strains is one of the most critical problems of modern medicine. As a result, a novel and most effective approaches for treating infection caused by multidrug-resistant bacteria are urgently required. In this context, one intriguing approach is to use bacteriophages (viruses that kill bacteria in the treatment of infection caused by drug-resistant bacteria. In the present study, the utility of lytic bacteriophages to rescue septicemic mice with multidrug-resistant (MDR P. aeruginosa infection was evaluated. MDR P. aeruginosa was used to induce septicemia in mice by intraperitoneal (i.p. injection of 10 7 CFU. The resulting bacteremia was fatal within 48 hrs. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR P. aeruginosa. A single i.p. injection of 3 x 10 9 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat-inactivated, they lose their ability to rescue the infected mice. Multidrug-resistant bacteria have

  5. Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates

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    Jin Jing

    2012-07-01

    Full Text Available Abstract Background Acinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization. Results Phage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min and a large burst size (200 PFU/cell. It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9 and at extreme temperatures (between 50°C and 60°C. ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages. Conclusion The phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent

  6. Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates.

    Science.gov (United States)

    Jin, Jing; Li, Zhen-Jiang; Wang, Shu-Wei; Wang, Shan-Mei; Huang, De-Hai; Li, Ya-Hui; Ma, Yun-Yun; Wang, Jin; Liu, Fang; Chen, Xiang-Dong; Li, Guang-Xing; Wang, Xiao-Ting; Wang, Zhong-Quan; Zhao, Guo-Qiang

    2012-07-28

    Acinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization. Phage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min) and a large burst size (200 PFU/cell). It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9) and at extreme temperatures (between 50°C and 60°C). ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages. The phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent; thus, it should be further investigated.

  7. Lytic bacteriophages reduce Escherichia coli O157: H7 on fresh cut lettuce introduced through cross-contamination.

    Science.gov (United States)

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm 2 ) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm 2 ). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm 2 ) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm 2 ) on day 0 compared with control treatments (4.10 log CFU/cm 2 ). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce.

  8. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii*

    OpenAIRE

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2015-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure ...

  9. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  10. Primary intraosseous atypical inflammatory meningioma presenting as a lytic skull lesion: Case report with review of literature.

    Science.gov (United States)

    Bohara, Sangita; Agarwal, Swapnil; Khurana, Nita; Pandey, P N

    2016-01-01

    Primary extradural meningiomas of the skull comprise 1% of all meningiomas, and lytic skull meningiomas are still rarer and are said to be more aggressive. We present a case of 38-year-old male with an extradural tumor which on histopathological examination showed features of inflammatory atypical meningioma (WHO Grade II). The intense inflammatory nature of osteolytic primary intraosseous meningioma has not been reported before. This entity deserves special mention because of the need for adjuvant therapy and proper follow-up.

  11. Human Cytotoxic T Lymphocytes Form Dysfunctional Immune Synapses with B Cells Characterized by Non-Polarized Lytic Granule Release

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    Anna Kabanova

    2016-04-01

    Full Text Available Suppression of the cytotoxic T cell (CTL immune response has been proposed as one mechanism for immune evasion in cancer. In this study, we have explored the underlying basis for CTL suppression in the context of B cell malignancies. We document that human B cells have an intrinsic ability to resist killing by freshly isolated cytotoxic T cells (CTLs, but are susceptible to lysis by IL-2 activated CTL blasts and CTLs isolated from immunotherapy-treated patients with chronic lymphocytic leukemia (CLL. Impaired killing was associated with the formation of dysfunctional non-lytic immune synapses characterized by the presence of defective linker for activation of T cells (LAT signaling and non-polarized release of the lytic granules transported by ADP-ribosylation factor-like protein 8 (Arl8. We propose that non-lytic degranulation of CTLs are a key regulatory mechanism of evasion through which B cells may interfere with the formation of functional immune synapses by CTLs.

  12. Cordycepin enhances Epstein-Barr virus lytic infection and Epstein-Barr virus-positive tumor treatment efficacy by doxorubicin.

    Science.gov (United States)

    Du, Yinping; Yu, Jieshi; Du, Li; Tang, Jun; Feng, Wen-Hai

    2016-07-01

    The consistent latent presence of Epstein-Barr virus (EBV) in tumor cells offers potential for virus-targeted therapies. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. In this study, we report that a natural small molecule compound, cordycepin, can induce lytic EBV infection in tumor cells. Subsequently, we demonstrate that cordycepin can enhance EBV reactivating capacity and EBV-positive tumor cell killing ability of low dose doxorubicin. The combination of cordycepin and doxorubicin phosphorylates CCAAT/enhancer binding protein β (C/EBPβ) through protein kinase C (PKC)-p38 mitogen activated protein kinases (p38 MAPK) signaling pathway, and C/EBPβ is required for the activation of lytic EBV infection. Most importantly, an in vivo experiment demonstrates that the combination of cordycepin and doxorubicin is more effective in inhibiting tumor growth in SCID mice than is doxorubicin alone. Our findings establish that cordycepin can enhance the efficacy of conventional chemotherapy for treatment of EBV-positive tumors. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

    Science.gov (United States)

    Tazzyman, Samuel J; Hall, Alex R

    2015-03-17

    The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

  14. Differential impact of lytic viruses on prokaryotic morphopopulations in a tropical estuarine system (Cochin estuary, India).

    Science.gov (United States)

    Jasna, Vijayan; Pradeep Ram, Angia Sriram; Parvathi, Ammini; Sime-Ngando, Telesphore

    2018-01-01

    Our understanding on the importance of viral lysis in the functioning of tropical estuarine ecosystem is limited. This study examines viral infection of prokaryotes and subsequent lysis of cells belonging to different morphotypes across a salinity gradient in monsoon driven estuarine ecosystem (Cochin estuary, India). High standing stock of viruses and prokaryotes accompanied by lytic infection rates in the euryhaline/mesohaline region of the estuary suggests salinity to have an influential role in driving interactions between prokaryotes and viruses. High prokaryotic mortality rates, up to 42% of prokaryote population in the pre-monsoon season is further substantiated by a high virus to prokaryote ratio (VPR), suggesting that maintenance of a high number of viruses is dependent on the most active fraction of bacterioplankton. Although myoviruses were the dominant viral morphotype (mean = 43%) throughout the study period, there was significant variation among prokaryotic morphotypes susceptible to viral infection. Among them, the viral infected short rod prokaryote morphotype with lower burst estimates (mean = 18 viruses prokaryote-1) was dominant (35%) in the dry seasons whereas a substantial increase in cocci forms (30%) infected by viruses with high burst size (mean = 31 viruses prokaryote-1) was evident during the monsoon season. Such preferential infections of prokaryotic morphopopulations with respect to seasons can have a strong and variable impact on the carbon and energy flow in this tropical ecosystem.

  15. The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii.

    Science.gov (United States)

    Crépin, Sébastien; Ottosen, Elizabeth N; Peters, Katharina; Smith, Sara N; Himpsl, Stephanie D; Vollmer, Waldemar; Mobley, Harry L T

    2018-06-08

    Acinetobacter baumannii has emerged as a leading nosocomial pathogen, infecting a wide range of anatomic sites including the respiratory tract and the bloodstream. In addition to being multi-drug resistant, little is known about the molecular basis of A. baumannii pathogenesis. To better understand A. baumannii virulence, a combination of a transposon-sequencing (TraDIS) screen and the neutropenic mouse model of bacteremia was used to identify the full set of fitness genes required during bloodstream infection. The lytic transglycosylase MltB was identified as a critical fitness factor. MltB cleaves the MurNAc-GlcNAc bond of peptidoglycan, which leads to cell wall remodeling. Here we show that MltB is part of a complex network connecting resistance to stresses, membrane homeostasis, biogenesis of pili and in vivo fitness. Indeed, inactivation of mltB not only impaired resistance to serum complement, cationic antimicrobial peptides and oxygen species, but also altered the cell envelope integrity, activated the envelope stress response, drastically reduced the number of pili at the cell surface and finally, significantly decreased colonization of both the bloodstream and the respiratory tract. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

  16. Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki.

    Directory of Open Access Journals (Sweden)

    Dann Turner

    Full Text Available Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA under the accession number LN890663.

  17. CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

    Science.gov (United States)

    Jain, Nidhi; Oswal, Neelam; Chawla, Amanpreet Singh; Agrawal, Tanvi; Biswas, Moanaro; Vrati, Sudhanshu; Rath, Satyajit; George, Anna; Bal, Vineeta; Medigeshi, Guruprasad R

    2017-02-01

    Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β-T cells (TCRβ-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

  18. CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

    Directory of Open Access Journals (Sweden)

    Nidhi Jain

    2017-02-01

    Full Text Available Following Japanese encephalitis virus (JEV infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β-T cells (TCRβ-null are highly susceptible and die over 10-18 day period as compared to the wild-type (WT mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB. Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

  19. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    Science.gov (United States)

    2011-01-01

    Background Several naturally occurring cationic antimicrobial peptides (CAPs), including bovine lactoferricin (LfcinB), display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS) on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS) on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS. PMID:21453492

  20. Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens.

    Science.gov (United States)

    Sulakvelidze, Alexander

    2013-10-01

    Bacteriophages (also called 'phages') are viruses that kill bacteria. They are arguably the oldest (3 billion years old, by some estimates) and most ubiquitous (total number estimated to be 10(30) -10(32) ) known organisms on Earth. Phages play a key role in maintaining microbial balance in every ecosystem where bacteria exist, and they are part of the normal microflora of all fresh, unprocessed foods. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention focused on using them to improve food safety. That approach, called 'phage biocontrol', typically includes three main types of applications: (i) using phages to treat domesticated livestock in order to reduce their intestinal colonization with, and shedding of, specific bacterial pathogens; (ii) treatments for decontaminating inanimate surfaces in food-processing facilities and other food establishments, so that foods processed on those surfaces are not cross-contaminated with the targeted pathogens; and (iii) post-harvest treatments involving direct applications of phages onto the harvested foods. This mini-review primarily focuses on the last type of intervention, which has been gaining the most momentum recently. Indeed, the results of recent studies dealing with improving food safety, and several recent regulatory approvals of various commercial phage preparations developed for post-harvest food safety applications, strongly support the idea that lytic phages may provide a safe, environmentally-friendly, and effective approach for significantly reducing contamination of various foods with foodborne bacterial pathogens. However, some important technical and nontechnical problems may need to be addressed before phage biocontrol protocols can become an integral part of routine food safety intervention strategies implemented by food industries in the USA. © 2013 Society of Chemical Industry.

  1. Antibacterial Efficacy of Lytic Bacteriophages against Antibiotic-Resistant Klebsiella Species

    Directory of Open Access Journals (Sweden)

    M. Khajeh Karamoddini

    2011-01-01

    Full Text Available Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran. Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation and solid (double-layer agar plate method; after 24 h of incubation phases. In each method, three different concentrations of bacteriophages (low: 107 PFU/mL were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  2. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    Directory of Open Access Journals (Sweden)

    Lindin Inger

    2011-03-01

    Full Text Available Abstract Background Several naturally occurring cationic antimicrobial peptides (CAPs, including bovine lactoferricin (LfcinB, display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS.

  3. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  4. Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

    Directory of Open Access Journals (Sweden)

    Laura L Quinn

    2014-08-01

    Full Text Available CD8+ T cell responses to Epstein-Barr virus (EBV lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE and some early (E antigens are more frequently observed than responses to epitopes of late (L expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE- and early (E-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L, interference by BILF1 increases with progression through lytic cycle (IElytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic

  5. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  6. Recurrent Plasmodium falciparum malaria infections in Kenyan children diminish T-cell immunity to Epstein Barr virus lytic but not latent antigens.

    Directory of Open Access Journals (Sweden)

    Cynthia J Snider

    Full Text Available Plasmodium falciparum malaria (Pf-malaria and Epstein Barr Virus (EBV infections coexist in children at risk for endemic Burkitt's lymphoma (eBL; yet studies have only glimpsed the cumulative effect of Pf-malaria on EBV-specific immunity. Using pooled EBV lytic and latent CD8+ T-cell epitope-peptides, IFN-γ ELISPOT responses were surveyed three times among children (10 months to 15 years in Kenya from 2002-2004. Prevalence ratios (PR and 95% confidence intervals (CI were estimated in association with Pf-malaria exposure, defined at the district-level (Kisumu: holoendemic; Nandi: hypoendemic and the individual-level. We observed a 46% decrease in positive EBV lytic antigen IFN-γ responses among 5-9 year olds residing in Kisumu compared to Nandi (PR: 0.54; 95% CI: 0.30-0.99. Individual-level analysis in Kisumu revealed further impairment of EBV lytic antigen responses among 5-9 year olds consistently infected with Pf-malaria compared to those never infected. There were no observed district- or individual-level differences between Pf-malaria exposure and EBV latent antigen IFN-γ response. The gradual decrease of EBV lytic antigen but not latent antigen IFN-γ responses after primary infection suggests a specific loss in immunological control over the lytic cycle in children residing in malaria holoendemic areas, further refining our understanding of eBL etiology.

  7. Vacuolar processing enzyme: an executor of plant cell death.

    Science.gov (United States)

    Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

    2005-08-01

    Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

  8. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    Science.gov (United States)

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Unprecedented evidence for high viral abundance and lytic activity in coral reef waters of the South Pacific Ocean

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    Jérôme P. Payet

    2014-09-01

    Full Text Available Despite nutrient-depleted conditions, coral reef waters harbor abundant and diverse microbes; as major agents of microbial mortality, viruses are likely to influence microbial processes in these ecosystems. However, little is known about marine viruses in these rapidly changing ecosystems. Here we examined spatial and short-term temporal variability in marine viral abundance and viral lytic activity across various reef habitats surrounding Moorea Island (French Polynesia in the South Pacific. Water samples were collected along 4 regional cross-reef transects and during a time-series in Opunohu Bay. Results revealed high viral abundance (range: 5.6 x 106 – 3.6 x 107 viruses ml-1 and lytic viral production (range: 1.5 x 109 – 9.2 x 1010 viruses l-1 d-1. Flow cytometry revealed that viral assemblages were composed of three subsets that each displayed distinct spatiotemporal relationships with nutrient concentrations and autotrophic and heterotrophic microbial abundances. The results highlight dynamic shifts in viral community structure and imply that each of these three subsets is ecologically important and likely to infect distinct microbial hosts in reef waters. Based on viral-reduction approach, we estimate that lytic viruses were responsible for the removal of ca. 24% to 367% of bacterial standing stock d-1 and the release of ca. 1.1 to 62 µg of organic carbon l-1 d-1 in reef waters. Overall, this work demonstrates the highly dynamic distribution of viruses and their critical roles in controlling microbial mortality and nutrient cycling in coral reef water ecosystems.

  10. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

    Science.gov (United States)

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii*

    Science.gov (United States)

    Nitzsche, Richard; Zagoriy, Vyacheslav; Lucius, Richard; Gupta, Nishith

    2016-01-01

    Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells. PMID:26518878

  12. Protoplast preparation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme

    International Nuclear Information System (INIS)

    Hassan Hamdani Mutaat; Mat Rasol Awang

    2004-01-01

    The objective of this study was to determine the optimum parameters of the factors influencing protoplast isolation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme from Trichoderma harzianurm. The study was conducted by manipulating the variables of the factors affecting protoplast isolation, such as age of mycelium culture, period for lysing of mycelium, concentration of lysing enzyme and concentration of osmotic stabilizer. The highest protoplast yield of 8.3 x 104 protoplast/ml was achieved when a 3-day P. sajor-caju mycelium, cultured statically, was incubated for 3 hours in a lytic mixture containing 7.5 mg/ml lysing enzyme and 1.2 M ammonium sulfate as osmotic stabilizer. This protoplast yield, however, is insufficient for regeneration and protoplast fusion works. (Author)

  13. De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia.

    Science.gov (United States)

    Sawtell, Nancy M; Thompson, Richard L

    2016-09-01

    The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.

  14. Volume of Lytic Vertebral Body Metastatic Disease Quantified Using Computed Tomography–Based Image Segmentation Predicts Fracture Risk After Spine Stereotactic Body Radiation Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Thibault, Isabelle [Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario (Canada); Department of Radiation Oncology, Centre Hospitalier de L' Universite de Québec–Université Laval, Quebec, Quebec (Canada); Whyne, Cari M. [Orthopaedic Biomechanics Laboratory, Sunnybrook Research Institute, Department of Surgery, University of Toronto, Toronto, Ontario (Canada); Zhou, Stephanie; Campbell, Mikki [Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario (Canada); Atenafu, Eshetu G. [Department of Biostatistics, University Health Network, University of Toronto, Toronto, Ontario (Canada); Myrehaug, Sten; Soliman, Hany; Lee, Young K. [Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario (Canada); Ebrahimi, Hamid [Orthopaedic Biomechanics Laboratory, Sunnybrook Research Institute, Department of Surgery, University of Toronto, Toronto, Ontario (Canada); Yee, Albert J.M. [Division of Orthopaedic Surgery, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario (Canada); Sahgal, Arjun, E-mail: arjun.sahgal@sunnybrook.ca [Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario (Canada)

    2017-01-01

    Purpose: To determine a threshold of vertebral body (VB) osteolytic or osteoblastic tumor involvement that would predict vertebral compression fracture (VCF) risk after stereotactic body radiation therapy (SBRT), using volumetric image-segmentation software. Methods and Materials: A computational semiautomated skeletal metastasis segmentation process refined in our laboratory was applied to the pretreatment planning CT scan of 100 vertebral segments in 55 patients treated with spine SBRT. Each VB was segmented and the percentage of lytic and/or blastic disease by volume determined. Results: The cumulative incidence of VCF at 3 and 12 months was 14.1% and 17.3%, respectively. The median follow-up was 7.3 months (range, 0.6-67.6 months). In all, 56% of segments were determined lytic, 23% blastic, and 21% mixed, according to clinical radiologic determination. Within these 3 clinical cohorts, the segmentation-determined mean percentages of lytic and blastic tumor were 8.9% and 6.0%, 0.2% and 26.9%, and 3.4% and 15.8% by volume, respectively. On the basis of the entire cohort (n=100), a significant association was observed for the osteolytic percentage measures and the occurrence of VCF (P<.001) but not for the osteoblastic measures. The most significant lytic disease threshold was observed at ≥11.6% (odds ratio 37.4, 95% confidence interval 9.4-148.9). On multivariable analysis, ≥11.6% lytic disease (P<.001), baseline VCF (P<.001), and SBRT with ≥20 Gy per fraction (P=.014) were predictive. Conclusions: Pretreatment lytic VB disease volumetric measures, independent of the blastic component, predict for SBRT-induced VCF. Larger-scale trials evaluating our software are planned to validate the results.

  15. Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Paspaliari, Dafni Katerina; Loose, Jennifer S. M.; Larsen, Marianne Halberg

    2015-01-01

    Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a ......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...... but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products...... (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium...

  16. The role of oxidative stress in EBV lytic reactivation, radioresistance and the potential preventive and therapeutic implications.

    Science.gov (United States)

    Hu, Jianmin; Li, Hongde; Luo, Xiangjian; Li, Yueshuo; Bode, Ann; Cao, Ya

    2017-11-01

    Epstein-Barr virus (EBV) is an important cancer causing virus. Cancer associated with EBV account for approximately 1.5% of all cancers, and represent 1.8% of all cancer deaths worldwide. EBV reactivation plays an important role in the development of EBV-related diseases and is closely related with patients' survival and clinical stages of EBV-related cancers. The therapy regarding to EBV-related cancers is very urgent, especially in endemic areas. Generating oxidative stress is a critical mechanism by which host cells defend against infection by virus. In addition, ROS-mediated oxidative stress plays a significant but paradoxical role acting as a "double-edged sword" to regulate cellular response to radiation, which is the main therapy strategy for EBV-related cancers, especially nasopharyngeal carcinoma. Therefore, in this review we primarily discuss the possible interplay among the oxidative stress, EBV lytic reactivation and radioresistance. Understanding the role of oxidative stress in EBV lytic reactivation and radioresistance will assist in the development of effective strategies for prevention and treatment of EBV-related cancers. © 2017 UICC.

  17. Effective inhibition of lytic development of bacteriophages λ, P1 and T4 by starvation of their host, Escherichia coli

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2007-02-01

    Full Text Available Abstract Background Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Results Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Conclusion Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

  18. Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV.

    Science.gov (United States)

    Church, Trenton Mel; Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

    2018-03-15

    Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81. IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in

  19. Comparison of MGIT and Myco/F lytic liquid-based blood culture systems for recovery of Mycobacterium tuberculosis from pleural fluid.

    Science.gov (United States)

    Harausz, Elizabeth; Lusiba, John Kafuluma; Nsereko, Mary; Johnson, John L; Toossi, Zahra; Ogwang, Sam; Boom, W Henry; Joloba, Moses L

    2015-04-01

    The specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery of Mycobacterium tuberculosis from pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively; P=0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

    Science.gov (United States)

    Filatova, Lyubov Y; Donovan, David M; Ishnazarova, Nadiya T; Foster-Frey, Juli A; Becker, Stephen C; Pugachev, Vladimir G; Balabushevich, Nadezda G; Dmitrieva, Natalia F; Klyachko, Natalia L

    2016-10-01

    A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal ( opt ) conditions: pH opt 6.0-10.0, t opt 20-30 °C, NaCl opt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

  1. Decalcified allograft in repair of lytic lesions of bone: A study to evolve bone bank in developing countries

    Directory of Open Access Journals (Sweden)

    Anil Kumar Gupta

    2016-01-01

    Full Text Available Background: The quest for ideal bone graft substitutes still haunts orthopedic researchers. The impetus for this search of newer bone substitutes is provided by mismatch between the demand and supply of autogenous bone grafts. Bone banking facilities such as deep frozen and freeze-dried allografts are not so widely available in most of the developing countries. To overcome the problem, we have used partially decalcified, ethanol preserved, and domestic refrigerator stored allografts which are economical and needs simple technology for procurement, preparation, and preservation. The aim of the study was to assess the radiological and functional outcome of the partially decalcified allograft (by weak hydrochloric acid in patients of benign lytic lesions of bone. Through this study, we have also tried to evolve, establish, and disseminate the concept of the bone bank. Materials and Methods: 42 cases of lytic lesions of bone who were treated by decalcified (by weak hydrochloric acid, ethanol preserved, allografts were included in this prospective study. The allograft was obtained from freshly amputated limbs or excised femoral heads during hip arthroplasties under strict aseptic conditions. The causes of lytic lesions were unicameral bone cyst ( n = 3, aneurysmal bone cyst ( n = 3, giant cell tumor ( n = 9, fibrous dysplasia ( n = 12, chondromyxoid fibroma, chondroma, nonossifying fibroma ( n = 1 each, tubercular osteomyelitis ( n = 7, and chronic pyogenic osteomyelitis ( n = 5. The cavity of the lesion was thoroughly curetted and compactly filled with matchstick sized allografts. Results: Quantitative assessment based on the criteria of Sethi et al. (1993 was done. There was complete assimilation in 27 cases, partial healing in 12 cases, and failure in 3 cases. Functional assessment was also done according to which there were 29 excellent results, 6 good, and 7 cases of failure (infection, recurrence, and nonunion of pathological fracture. We

  2. Latent and lytic HHV-8 mRNA expression in PBMCs and Kaposi's sarcoma skin biopsies of AIDS Kaposi's sarcoma patients

    NARCIS (Netherlands)

    Polstra, Abeltje M.; Goudsmit, Jaap; Cornelissen, Marion

    2003-01-01

    Human herpes virus 8 (HHV-8) is associated with all clinical forms of Kaposi's sarcoma. HHV-8 DNA is present in Kaposi's sarcoma biopsies and is observed regularly in saliva and less consistently in blood of Kaposi's sarcoma patients. The expression pattern of latent (ORF 73) and lytic (vGCR,

  3. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  4. Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

    Science.gov (United States)

    Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

    2010-01-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

  5. Combined Inhibition of the BMP pathway and the RANK-RANKL axis in a Mixed Lytic/blastic Prostate Cancer Lesion

    Science.gov (United States)

    Virk, Mandeep S.; Alaee, Farhang; Petrigliano, Frank A.; Sugiyama, Osamu; Chatziioannou, Arion F.; Stout, David; Dougall, William C.; Lieberman, Jay R.

    2010-01-01

    The purpose of this study was to investigate the influence of combined inhibition of RANKL (receptor activator of nuclear factor kappa-B ligand) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hind limb tumor size, 18F-Fluorodeoxyglucose and 18F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone. PMID:21073986

  6. Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

    Science.gov (United States)

    Biziulevicius, Gediminas A

    2006-01-01

    Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

  7. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  8. Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.

    Science.gov (United States)

    Sun, Fubao Fuebiol; Hong, Jiapeng; Hu, Jinguang; Saddler, Jack N; Fang, Xu; Zhang, Zhenyu; Shen, Song

    2015-11-01

    The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Abortive lytic Epstein–Barr virus replication in tonsil-B lymphocytes in infectious mononucleosis and a subset of the chronic fatigue syndrome

    Directory of Open Access Journals (Sweden)

    Lerner AM

    2012-11-01

    Full Text Available A Martin Lerner,1 Safedin Beqaj21Department of Medicine, Oakland University William Beaumont School of Medicine, Rochester, MI, USA; 2Pathology Inc, Torrance, CA, USAAbstract: A systematic 2001–2007 review of 142 chronic fatigue syndrome (CFS patients identified 106 CFS patients with elevated serum IgG antibodies to the herpesviruses Epstein–Barr virus (EBV, cytomegalovirus, or human herpesvirus (HHV 6 in single or multiple infections, with no other co-infections detected. We named these 106 patients group-A CFS. Eighty-six of these 106 group-A CFS patients (81% had elevated EBV early antibody, early antigen (diffuse, serum titers. A small group of six patients in the group-A EBV subset of CFS, additionally, had repetitive elevated-serum titers of antibody to the early lytic replication-encoded proteins, EBV dUTPase, and EBV DNA polymerase. The presence of these serum antibodies to EBV dUTPase and EBV DNA polymerase indicated EBV abortive lytic replication in these 6 CFS patients. None of 20 random control people (age- and sex-matched, with blood drawn at a commercial laboratory had elevated serum titers of antibody to EBV dUTPase or EBV DNA polymerase (P < 0.01. This finding needs verification in a larger group of EBV CFS subset patients, but if corroborated, it may represent a molecular marker for diagnosing the EBV subset of CFS. We review evidence that EBV abortive lytic replication with unassembled viral proteins in the blood may be the same in infectious mononucleosis (IM and a subset of CFS. EBV-abortive lytic replication in tonsil plasma cells is dominant in IM. No complete lytic virion is in the blood of IM or CFS patients. Complications of CFS and IM include cardiomyopathy and encephalopathy. Circulating abortive lytic-encoded EBV proteins (eg, EBV dUTPase, EBV DNA polymerase, and others may be common to IM and CFS. The intensity and duration of the circulating EBV-encoded proteins might differentiate the IM and EBV subsets of CFS

  10. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  11. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  12. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  13. Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle.

    Science.gov (United States)

    Horcajo, P; Xia, D; Randle, N; Collantes-Fernández, E; Wastling, J; Ortega-Mora, L M; Regidor-Cerrillo, J

    2017-11-14

    Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study

  14. Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

    Science.gov (United States)

    Leatham, Gary F.

    1985-01-01

    Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

  15. Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types.

    Science.gov (United States)

    Solovieva, Ekaterina V; Myakinina, Vera P; Kislichkina, Angelina A; Krasilnikova, Valentina M; Verevkin, Vladimir V; Mochalov, Vladimir V; Lev, Anastasia I; Fursova, Nadezhda K; Volozhantsev, Nikolay V

    2018-01-02

    Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395-44,623bp including direct terminal repeats of 180-246 bp. The G + C contents are 52.3-54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  17. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  18. Targeted enzyme prodrug therapies.

    Science.gov (United States)

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  19. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  20. A study by nitrogen-15 nuclear magnetic resonance spectroscopy of the state of histidine in the catalytic triad of α-lytic protease

    International Nuclear Information System (INIS)

    Bachovchin, W.W.; Roberts, J.D.

    1978-01-01

    The ionization behaviour of the histidine of the catalytic triad of α-lytic protease using N-15 NMR spectroscopy is studied. This technique is especially informative about the protonation, hydrogen-bond formation, and tautomeric equilibrium of imidazole rings. The efficient and specific incorporation of N-15 labelled histidine into α-lytic protease was achieved by inducing and isolating an auxotroph of myxobacter 495 for which histidine is an essential amino acid. The results show that histidine of the catalytic triad of α-lytic protease appears to have a base strength which is essentially normal for an imidazole derivative but, in the pH range where the enzymatic activity is high, the histidine tautomer is favoured with the hydrogen located on N3 (π), as the result of hydrogen bonding to the asparate anion and possible the serine hydroxyl. Thus, the N-15 NMR shifts support the general geometry postulated for the ''charge-relay'' mechanism but not the idea of an unusually weakly basic histidine or an unusually strongly basic asparate carboxylate anion. (A.G.)

  1. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-03-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers.

  2. Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

    Science.gov (United States)

    Yue, Yang; Yu, Huahua; Li, Rongfeng; Xing, Ronge; Liu, Song; Li, Kecheng; Wang, Xueqin; Chen, Xiaolin; Li, Pengcheng

    2017-01-26

    Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii , through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N . nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

  3. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    International Nuclear Information System (INIS)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-01-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers

  4. pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

    Science.gov (United States)

    Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

    2018-05-01

    The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. In vitro characterization and in vivo properties of Salmonellae lytic bacteriophages isolated from free-range layers

    Directory of Open Access Journals (Sweden)

    L Fiorentin

    2004-06-01

    Full Text Available Occurrence of food poisoning related to Salmonella-contaminated eggs and chicken meat has been frequent in humans. Salmonella Enteritidis (SE and Salmonella Typhimurium (ST are included among the most important paratyphoid salmonellae associated with chicken meat and eggs. Elimination of Salmonella at the pre-harvest stage can play a significant role in preventing the introduction of this pathogen into the food chain and consequently in the reduction of food poisoning in humans. Bactericidal bacteriophages may provide a natural, nontoxic, feasible and non-expensive component of the multi-factorial approach for a pre-harvest control of Salmonella in poultry. Five bacteriophages lytic for SE PT4 and ST were obtained from 107 samples of feces of free-range layers in Brazil. All bacteriophages were characterized in vitro and in vivo, showing head and tail morphology and dsDNA as nucleic acids. Results of "in vivo" studies suggested that bacteriophages do not remain in Salmonella-free birds longer than one day, whereas they multiply in Salmonella-infected birds for longer periods. Besides, selection for phage-resistant SE PT4 did not seem to occur in the short term. Isolated bacteriophages will be investigated for their potential for pre-harvest biocontrol of SE PT4 in poultry.

  6. The role of staphylococci in subclinical mastitis of cows and lytic phage isolation against to Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Aliye Gulmez Saglam

    2017-12-01

    Full Text Available Aim: This study was conducted to determine the role of Staphylococcus in the formation of subclinical mastitis in cows and to isolate the phage against isolated Staphylococcus aureus strains. Materials and Methods: In this study, 400 milk cows were screened by California Mastitis Test (CMT for subclinical mastitis and 235 udders of 96 cows, which were determined to be positive, were evaluated for Staphylococcus. Milk samples were evaluated using conventional and molecular methods. In addition, phage isolation studies were performed against S. aureus strains causing mastitis. Results: At the result of cultural examination, of 235 milk samples that were found as positive for mastitis by CMT, a total of 117 (49.7% Staphylococcus spp. were isolated as a distribution of 74 (63.24% coagulase-positive staphylococci and 43 (36.75% coagulase-negative staphylococci. Of these isolates, 76 (64.95% were characterized as S. aureus both conventional and molecular techniques. Lytic bacteriophages against two S. aureus strains which were isolated from mastitic milk samples were obtained from wastewater samples. Conclusion: The results of this study show that a significant portion of subclinical mastitis was formed by staphylococci. In addition, phage isolation against S. aureus strains isolated can be considered as one of the steps to be applied in the prophylaxis and treatment of such infections.

  7. Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection

    Directory of Open Access Journals (Sweden)

    Janice K. P. Lam

    2018-03-01

    Full Text Available Long term carriers were shown to generate robust polyfunctional T cell (PFC responses against lytic and latent antigens of Epstein-Barr virus (EBV. However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM] and asymptomatic (AS EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6–12 months. In conclusion, EBV antigen-specific CD4+ and CD

  8. Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis.

    Science.gov (United States)

    Kim, Hyoji; Choi, Hoyun; Lee, Suk Kyeong

    2016-02-01

    Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the

  9. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J J; Brand, J C

    1980-01-01

    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  10. Indicators: Sediment Enzymes

    Science.gov (United States)

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  11. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzymes are biocatalytic protein molecules that enhance the rates of ... to physical forces (hydrogen bonds, hydrophobic 1, electrostatic and Van der ... conformation. In 1995 ... surface against 14.7% in Klenow poll (some of the hydrophobic.

  12. Overproduction of ligninolytic enzymes

    Science.gov (United States)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  13. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  14. Reduction of Salmonella on chicken meat and chicken skin by combined or sequential application of lytic bacteriophage with chemical antimicrobials.

    Science.gov (United States)

    Sukumaran, Anuraj T; Nannapaneni, Rama; Kiess, Aaron; Sharma, Chander Shekhar

    2015-08-17

    The effectiveness of recently approved Salmonella lytic bacteriophage preparation (SalmoFresh™) in reducing Salmonella in vitro and on chicken breast fillets was examined in combination with lauric arginate (LAE) or cetylpyridinium chloride (CPC). In another experiment, a sequential spray application of this bacteriophage (phage) solution on Salmonella inoculated chicken skin after a 20s dip in chemical antimicrobials (LAE, CPC, peracetic acid, or chlorine) was also examined in reducing Salmonella counts on chicken skin. The application of phage in combination with CPC or LAE reduced S. Typhimurium, S. Heidelberg, and S. Enteritidis up to 5 log units in vitro at 4 °C. On chicken breast fillets, phage in combination with CPC or LAE resulted in significant (p<0.05) reductions of Salmonella ranging from 0.5 to 1.3 log CFU/g as compared to control up to 7 days of refrigerated storage. When phage was applied sequentially with chemical antimicrobials, all the treatments resulted in significant reductions of Salmonella. The application of chlorine (30 ppm) and PAA (400 ppm) followed by phage spray (10(9)PFU/ml) resulted in highest Salmonella reductions of 1.6-1.7 and 2.2-2.5l og CFU/cm(2), respectively. In conclusion, the surface applications of phage in combination with LAE or CPC significantly reduced Salmonella counts on chicken breast fillets. However, higher reductions in Salmonella counts were achieved on chicken skin by the sequential application of chemical antimicrobials followed by phage spray. The sequential application of chlorine, PAA, and phage can provide additional hurdles to reduce Salmonella on fresh poultry carcasses or cut up parts. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms’ Lytic Activity Using Mass Spectrometry and Zymography

    Science.gov (United States)

    Yue, Yang; Yu, Huahua; Li, Rongfeng; Xing, Ronge; Liu, Song; Li, Kecheng; Wang, Xueqin; Chen, Xiaolin; Li, Pengcheng

    2017-01-01

    Background: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. Methods: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Results: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14–18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. Conclusion: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom. PMID:28134758

  16. Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection

    Directory of Open Access Journals (Sweden)

    Jennifer S. Lee

    2016-01-01

    Full Text Available Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1 genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3 lysine 9-trimethylation (H3K9me3 and lysine 27-trimethylation (H3K27me3 during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms.

  17. Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms’ Lytic Activity Using Mass Spectrometry and Zymography

    Directory of Open Access Journals (Sweden)

    Yang Yue

    2017-01-01

    Full Text Available Background: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. Methods: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS. The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Results: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14–18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. Conclusion: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

  18. Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth

    KAUST Repository

    Araujo, Paula Alexandra Da Silva; Machado, Idalina; Meireles, Ana; Leiknes, TorOve; Mergulhã o, Filipe; Melo, Luí s F.; Simõ es, Manuel

    2017-01-01

    Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB−protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrowth significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.

  19. Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth

    KAUST Repository

    Araujo, Paula Alexandra Da Silva

    2017-03-01

    Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB−protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrowth significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.

  20. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  1. Bone marrow edema pattern identification in patients with lytic bone lesions using digital subtraction angiography-like bone subtraction on large-area detector computed tomography.

    Science.gov (United States)

    Gondim Teixeira, Pedro Augusto; Hossu, Gabriela; Lecocq, Sophie; Razeto, Marco; Louis, Matthias; Blum, Alain

    2014-03-01

    The objective of this study was to evaluate the performance of digital subtraction angiography (DSA)-like bone subtraction with 2 different registration methods for the identification of bone marrow edema pattern (BMEP) in patients with lytic bone lesions, using magnetic resonance imaging as the criterion standard. Fifty-five patients with a lytic bone lesion were included in this prospective study with approval from the ethics committee. All patients underwent magnetic resonance imaging and low-dose computed tomographic (CT) perfusion after signing an informed consent. Two CT volumes were used for bone subtraction, which was performed with 2 different algorithms (rigid and nonrigid). Enhancement at the nonlytic bone marrow was considered as a sign of BMEP. Two readers evaluated the images blindly. The presence of BMEP on bone-subtracted CT images was evaluated subjectively and quantitatively. Image quality was assessed. Magnetic resonance imaging was used as the criterion standard. Using a rigid registration method, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of CT with DSA-like bone subtraction BMEP was 77%, 100%, 100%, 68%, and 85%, respectively. The interobserver agreement was good (κ, 0.782). Image quality was better using a nonrigid registration. With this algorithm, artifacts interfered with image interpretation in only 5% of cases. However, there was a noticeable drop in sensitivity and negative predictive value when a nonrigid algorithm was used: 56% and 52%, respectively. The interobserver agreement was average with a nonrigid subtraction algorithm. Computed tomography with DSA-like bone subtraction is sensitive and highly specific for the identification of BMEP associated with lytic bone lesions. Rigid registering should be preferred, but nonrigid algorithms can be used as a second option when artifacts interfere with image interpretation.

  2. Cellular corepressor TLE2 inhibits replication-and-transcription- activator-mediated transactivation and lytic reactivation of Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    He, Zhiheng; Liu, Yunhua; Liang, Deguang; Wang, Zhuo; Robertson, Erle S; Lan, Ke

    2010-02-01

    Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-Jkappa, Ap-1, C/EBP-alpha, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-Jkappa has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

  3. Matrix Metalloproteinase Enzyme Family

    Directory of Open Access Journals (Sweden)

    Ozlem Goruroglu Ozturk

    2013-04-01

    Full Text Available Matrix metalloproteinases play an important role in many biological processes such as embriogenesis, tissue remodeling, wound healing, and angiogenesis, and in some pathological conditions such as atherosclerosis, arthritis and cancer. Currently, 24 genes have been identified in humans that encode different groups of matrix metalloproteinase enzymes. This review discuss the members of the matrix metalloproteinase family and their substrate specificity, structure, function and the regulation of their enzyme activity by tissue inhibitors. [Archives Medical Review Journal 2013; 22(2.000: 209-220

  4. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  5. Magnetically responsive enzyme powders

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Šafařík, Ivo

    2015-01-01

    Roč. 380, APR 2015 (2015), s. 197-200 ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 2.357, year: 2015

  6. Enzyme with rhamnogalacturonase activity.

    NARCIS (Netherlands)

    Kofod, L.V.; Andersen, L.N.; Dalboge, H.; Kauppinen, M.S.; Christgau, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A.G.J.; Schols, H.A.

    1998-01-01

    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet

  7. Implantable enzyme amperometric biosensors.

    Science.gov (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Advances in enzyme bioelectrochemistry

    Directory of Open Access Journals (Sweden)

    ANDRESSA R. PEREIRA

    Full Text Available ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

  9. Cold-Adapted Enzymes

    Science.gov (United States)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  10. Embedded enzymes catalyse capture

    Science.gov (United States)

    Kentish, Sandra

    2018-05-01

    Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

  11. Photoperiodism and Enzyme Activity

    Science.gov (United States)

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  12. ISFET based enzyme sensors

    NARCIS (Netherlands)

    van der Schoot, Bart H.; Bergveld, Piet

    1987-01-01

    This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the

  13. The Enzyme Function Initiative†

    Science.gov (United States)

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  14. The Enzyme Function Initiative.

    Science.gov (United States)

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

    2011-11-22

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.

  15. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

    Directory of Open Access Journals (Sweden)

    Libera Latino

    Full Text Available A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31 is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10 with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

  16. Role of defective Oct-2 and OCA-B expression in immunoglobulin production and Kaposi's sarcoma-associated herpesvirus lytic reactivation in primary effusion lymphoma.

    Science.gov (United States)

    Di Bartolo, Daniel L; Hyjek, Elizabeth; Keller, Shannon; Guasparri, Ilaria; Deng, Hongyu; Sun, Ren; Chadburn, Amy; Knowles, Daniel M; Cesarman, Ethel

    2009-05-01

    Primary effusion lymphoma (PEL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Despite having a genotype and gene expression signature of highly differentiated B cells, PEL does not usually express surface or cytoplasmic immunoglobulin (Ig). We show the lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this defect in Ig production. Like Ig genes, ORF50, the key regulator of the switch from latency to lytic reactivation, contains an octamer motif within its promoter. We therefore examined the impact of Oct-2 and OCA-B on ORF50 activation. The binding of Oct-1 to the ORF50 promoter has been shown to significantly enhance ORF50 transactivation. We found that Oct-2, on the other hand, inhibited ORF50 expression and consequently lytic reactivation by competing with Oct-1 for the octamer motif in the ORF50 promoter. Our data suggest that Oct-2 downregulation in infected cells would be favorable to KSHV in allowing for efficient viral reactivation.

  17. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    Science.gov (United States)

    Husseneder, Claudia; Donaldson, Jennifer R; Foil, Lane D

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.

  18. Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

    Science.gov (United States)

    Otero, Fátima; Tamayo, María; Santiso, Rebeca; Gosálvez, Jaime; Bou, Germán; Fernández, José Luis

    2017-04-01

    A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

  19. NRSA enzyme decomposition model data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  20. Cellulase enzyme and biomass utilization

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... human population grows and economic development. However, the current .... conditions and the production cost of the related enzyme system. Therefore ... Given the importance of this enzyme to these so many industries,.

  1. The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

    Directory of Open Access Journals (Sweden)

    Rebecca C Robey

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is causally related to Kaposi's sarcoma (KS, the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs. We used these to transduce monocyte-derived dendritic cells (moDCs isolated from 14 KSHV-seropositive (12 HIV-positive and 7 KSHV-seronegative (4 HIV-positive individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle. Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1] were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37] was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1] was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

  2. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel

    2017-01-01

    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

  3. Characterising Complex Enzyme Reaction Data.

    Directory of Open Access Journals (Sweden)

    Handan Melike Dönertaş

    Full Text Available The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG. Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

  4. Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol.

    Directory of Open Access Journals (Sweden)

    Zoe A Dyson

    Full Text Available Nine bacteriophages (phages infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

  5. The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.

    Directory of Open Access Journals (Sweden)

    Mohammed Almuhayawi

    Full Text Available Detection and identification of anaerobic bacteria in blood cultures (BC is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux, BACTEC-Plus and -Lytic (Becton Dickinson BioSciences BC bottles in detection and time to detection (TTD of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94% than BacT/ALERT FN Plus (80/100, 80% (p<0.01 in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001. The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h, BACTEC Plus (27 h and finally BacT/ALERT FN Plus (38 h bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76% BacT/ALERT FN, 51/67 (76% BacT/ALERT FN Plus, 53/67 (79% BACTEC Plus and 50/67 (75% BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.

  6. PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

    Science.gov (United States)

    Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang

    2018-04-18

    The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  7. Co-therapy using lytic bacteriophage and linezolid: effective treatment in eliminating methicillin resistant Staphylococcus aureus (MRSA from diabetic foot infections.

    Directory of Open Access Journals (Sweden)

    Sanjay Chhibber

    Full Text Available BACKGROUND: Staphylococcus aureus remains the predominant pathogen in diabetic foot infections and prevalence of methicillin resistant S.aureus (MRSA strains further complicates the situation. The incidence of MRSA in infected foot ulcers is 15-30% and there is an alarming trend for its increase in many countries. Diabetes acts as an immunosuppressive state decreasing the overall immune functioning of body and to worsen the situation, wounds inflicted with drug resistant strains represent a morbid combination in diabetic patients. Foot infections caused by MRSA are associated with an increased risk of amputations, increased hospital stay, increased expenses and higher infection-related mortality. Hence, newer, safer and effective treatment strategies are required for treating MRSA mediated diabetic foot infections. The present study focuses on the use of lytic bacteriophage in combination with linezolid as an effective treatment strategy against foot infection in diabetic population. METHODOLOGY: Acute hindpaw infection with S.aureus ATCC 43300 was established in alloxan induced diabetic BALB/c mice. Therapeutic efficacy of a well characterized broad host range lytic bacteriophage, MR-10 was evaluated alone as well as in combination with linezolid in resolving the course of hindpaw foot infection in diabetic mice. The process of wound healing was also investigated. RESULTS AND CONCLUSIONS: A single administration of phage exhibited efficacy similar to linezolid in resolving the course of hindpaw infection in diabetic animals. However, combination therapy using both the agents was much more effective in arresting the entire infection process (bacterial load, lesion score, foot myeloperoxidase activity and histopathological analysis. The entire process of tissue healing was also hastened. Use of combined agents has been known to decrease the frequency of emergence of resistant mutants, hence this approach can serve as an effective strategy in

  8. Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Małgorzata Tokarska-Rodak

    2015-11-01

    Full Text Available Background: Atomic force microscopy (AFM is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. Material and methods: In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young’s modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker.Results/Conclusion: The average value of flexibility of spirochetes’ surface expressed by Young’s modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young’s modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

  9. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  10. Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

    Science.gov (United States)

    Hickey, Rita M.; Ross, R. Paul; Hill, Colin

    2004-01-01

    This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems. PMID:15006800

  11. Interaction and modulation of two antagonistic cell wall enzymes of mycobacteria.

    Directory of Open Access Journals (Sweden)

    Erik C Hett

    2010-07-01

    Full Text Available Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB, a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA, an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1, as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein-protein interactions between enzymes with antagonistic functions.

  12. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  13. DGAT enzymes and triacylglycerol biosynthesis

    Science.gov (United States)

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

  14. Enzyme stabilization for pesticide degradation

    Energy Technology Data Exchange (ETDEWEB)

    Rivers, D.B.; Frazer, F.R. III; Mason, D.W.; Tice, T.R.

    1988-01-01

    Enzymes offer inherent advantages and limitations as active components of formulations used to decontaminate soil and equipment contaminated with toxic materials such as pesticides. Because of the catalytic nature of enzymes, each molecule of enzyme has the potential to destroy countless molecules of a contaminating toxic compound. This degradation takes place under mild environmental conditions of pH, temperature, pressure, and solvent. The basic limitation of enzymes is their degree of stability during storage and application conditions. Stabilizing methods such as the use of additives, covalent crosslinking, covalent attachment, gel entrapment, and microencapsulation have been directed developing an enzyme preparation that is stable under extremes of pH, temperature, and exposure to organic solvents. Initial studies were conducted using the model enzymes subtilisin and horseradish peroxidase.

  15. Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

    NARCIS (Netherlands)

    van Noorden, Cornelis J. F.

    2002-01-01

    Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

  16. Enzyme Mimics: Advances and Applications.

    Science.gov (United States)

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. [The rise of enzyme engineering in China].

    Science.gov (United States)

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

  18. Enzyme structure, enzyme function and allozyme diversity in ...

    African Journals Online (AJOL)

    In estimates of population genetic diversity based on allozyme heterozygosity, some enzymes are regularly more variable than others. Evolutionary theory suggests that functionally less important molecules, or parts of molecules, evolve more rapidly than more important ones; the latter enzymes should then theoretically be ...

  19. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    Science.gov (United States)

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

  20. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  1. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care....... However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...

  2. Enzymes in Human Milk.

    Science.gov (United States)

    Dallas, David C; German, J Bruce

    2017-01-01

    Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system. © 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.

  3. Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

    Directory of Open Access Journals (Sweden)

    Richard J Kraus

    2017-06-01

    Full Text Available When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs. We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV. Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK and gingival epithelial (hGET cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for

  4. Digestive enzymes of some earthworms.

    Science.gov (United States)

    Mishra, P C; Dash, M C

    1980-10-15

    4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

  5. Photoreactivating enzyme from Escherichia coli

    International Nuclear Information System (INIS)

    Snapka, R.M.; Fuselier, C.O.

    1977-01-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm. (author)

  6. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  7. Photoreactivating enzyme from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Snapka, R M; Fuselier, C O [California Univ., Irvine (USA)

    1977-05-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm.

  8. Development of thermophilic tailor-made enzyme mixtures for the bioconversion of agricultural and forest residues

    Directory of Open Access Journals (Sweden)

    Anthi eKarnaouri

    2016-02-01

    Full Text Available Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5 and MtEG7, in the presence of other four accessory enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a and β-glucosidase MtBGL3, were tested as a 9-component cocktail against one model substrate (phosphoric acid swollen cellulose and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch and spruce biomass, as forest residues. Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC, high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch. Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that

  9. BAKERY ENZYMES IN CEREAL TECHNOLOGIES

    Directory of Open Access Journals (Sweden)

    Václav Koman

    2012-10-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

  10. [Automated analyzer of enzyme immunoassay].

    Science.gov (United States)

    Osawa, S

    1995-09-01

    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

  11. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    are affected (in a positive or negative way) by the presence of the other enzymes and compounds in the media. In this thesis the concept of multi-enzyme in-pot term is adopted for processes that are carried out by the combination of enzymes in a single reactor and implemented at pilot or industrial scale...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective....... In this way the model parameters that drives the main dynamic behavior can be identified and thus a better understanding of this type of processes. In order to develop, test and verify the methodology, three case studies were selected, specifically the bi-enzyme process for the production of lactobionic acid...

  12. PIXE analysis of Zn enzymes

    International Nuclear Information System (INIS)

    Solis, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J.L.; Romero, I.; Celis, H.

    1999-01-01

    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillum rubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy

  13. GRE Enzymes for Vector Analysis

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  14. Watching Individual Enzymes at Work

    Science.gov (United States)

    Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

    Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

  15. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  16. Lytic process studies on anaerobic digestion of organic wastes. Etude des activites lytiques intervenant au cours de la digestion anaerobie des dechets organiques; Rapport final

    Energy Technology Data Exchange (ETDEWEB)

    Durecu, S.; Thauront, J. (PEC Engineering, 95 - Cergy Pontoise (France). Service de Recherche et Developpement); Festino, C.; Aubart, C.; Reisinger, O. (Nancy-1 Univ., 54 - Vandoeuvre-les-Nancy (France). Lab. d' Ecologie Microbienne)

    1990-01-01

    To improve anaerobic digestion of pig manure, solubilization of the solid fraction was studied as the rate limiting step in the biomethanation process in an experimental completely mixed digester. The performance of conventional digesters should anaerobic microflora were inefficient in degrading complex biopolymers such as plant fibers. For pectin or cellulose, the use of digestible co-substrates accelerated methanation by increasing the yield of methane and a doubling of the apparent first order solubilization rate constant (Kp = 0.090/d). Lignin should methanation by decreasing methane yield and reducing the rate constant (Kp = 0.035/d). This inhibition was unrelated to volatile fatty acid accumulation. Nine strains of pectinolytic and/or cellulolytic bacteria were isolated. Chitin, a structural constituent of many final species, was effectively solubilized dining anaerobic digestion of pig manure. Seven strains of chitinolytic bacteria were isolated by high chitnese activity. The mycolytic power of fermenting manure processes acting through lytic microflora has been shown to be an effective antagonist of soil borne phytopathogenic fungi, as well as a fertilizer. In greenhouse trails, this compiled fraction demonstrated its ability to control flux unit. Keratin enhanced methane production, and increased H{sub 2}S nearly six-fold. Bacterial strains able to solubilize keratin were also used in autoclawed feather meal to extract the amino acids. (KJD)

  17. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida--evidence for close relationship to the T7 group

    International Nuclear Information System (INIS)

    Kovalyova, Irina V.; Kropinski, Andrew M.

    2003-01-01

    The genome of the lytic Pseudomonas putida bacteriophage gh-1 is linear double-stranded DNA containing 37,359 bp with 216-bp direct terminal repeats. Like other members of the T7 group, the gh-1 genome contains regions of high homology to T7 interspersed with nonhomologous regions that contain small open reading frames of unknown function. The genome shares 31 genes in common with other members of the T7 group, including RNA polymerase, and an additional 12 unique putative genes. A major difference between gh-1 and other members of this group is the absence of any open reading frames between the left direct terminal repeat and gene 1. Sequence analysis of the gh-1 genome also revealed the presence of 10 putative phage promoters with a consensus sequence similar to the promoters of T3 and phiYeO3-12 (consensus: TAAAAACCCTCACTRTGGCHSCM). P. putida mutants resistant to gh-1 were demonstrated to have an altered lipopolysaccharide structure, indicating that members of this group use lipopolysaccharide as their cellular receptor

  18. Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Gwan-Han Shen

    Full Text Available AIMS: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy. METHODS AND RESULTS: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8 CFU ml(-1 to 10(3 CFU ml(-1 in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314 as a cocktail could lyse all genotype-varying XDRAB isolates. CONCLUSION: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

  19. DGAT enzymes and triacylglycerol biosynthesis

    OpenAIRE

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, ...

  20. Enzymes: principles and biotechnological applications

    Science.gov (United States)

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  1. de novo computational enzyme design.

    Science.gov (United States)

    Zanghellini, Alexandre

    2014-10-01

    Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  3. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    Science.gov (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  4. Rethinking fundamentals of enzyme action.

    Science.gov (United States)

    Northrop, D B

    1999-01-01

    Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

  5. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  6. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  7. Substrate mediated enzyme prodrug therapy

    DEFF Research Database (Denmark)

    Fejerskov, Betina; Jarlstad Olesen, Morten T; Zelikin, Alexander N

    2017-01-01

    Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug administra......Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug...

  8. Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

    Science.gov (United States)

    Mikitas, Olga V; Ivin, Yuri Y; Golyshev, Sergey A; Povarova, Natalia V; Galkina, Svetlana I; Pletjushkina, Olga Y; Nadezhdina, Elena S; Gmyl, Anatoly P; Agol, Vadim I

    2012-05-01

    Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

  9. UV irradiation of Shigella Dysenteriae induced the transformation and excision of a presumed integrated lysogenic prophage Shd-4L10 into a lytic phase

    International Nuclear Information System (INIS)

    Rodrigues, F. K.; Addy, B.; Armah, G.; Fobil, J.; Steiner-Asiedu, M.; Efavi, J.

    2012-01-01

    Repeated exposures of Shigella dysenteriae strain A to ultra-violet radiation (253.7 nm) with intervening outgrowth of survivors gave rise to clear bacteriophage plaques. Isolation, propagation and partial purification of the new Shd-4LI0 phages showed that they are similar in morphology to the Myxobacteriaphage Mx-4 described earlier. The new phages retained the general characteristics of S. dystenteriae phageShd-4L3, including serological properties and phage typing. It is suggested that ultra-violet irradiation may have played a role in the transformation and excision of the presumed lysogen of S. dysenteriaestrain A into a lytic phase. PhageShd-4LI0 was subsequently partially characterized. It has a density of 1.61, a DNA: protein ratio of 0.42 and thus a cryptogram of D/2:54.3/32.5:X/X: B/O. The phage was further characterised by fractionation of its protein using SDS-polyacrilamide gel electrophoresis. DNA extracted from phages was hydrolysed with restriction endonuclease R., EcoR1. The restriction fragments were catalogued and their apparent molecular weights calculated from electrophoresis gels calibrated with fragments from DNA of coliphageλ λ. From the total fragments obtained with nuclease R., EcoR1, the apparent minimum molecular weight of phage Shd-4LI0DNA was found to be 54.3 x 10 6 Daltons. The molecular weight of the phage DNA was also calculated from measurements of contour length of purified DNA samples, using the formula MW = 1.97 x 10 10 1/(magnification), where 1 is the measured length of DNA in centimetres). The very close relatedness with phage Shd-4L3 was confirmed by these techniques. (au)

  10. Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance.

    Directory of Open Access Journals (Sweden)

    Alexander G Bulakhov

    Full Text Available Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO displaying a synergism with cellulases.Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL, and eglIV, encoding LPMO (formerly endoglucanase IV from Trichoderma reesei (TrLPMO, were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3 varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples. The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10-43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable

  11. Thermodynamics of Enzyme-Catalyzed Reactions Database

    Science.gov (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  12. Curious Cases of the Enzymes.

    Science.gov (United States)

    Ulusu, Nuriye Nuray

    2015-07-01

    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the 'trial and error' methodology of the ancients is now replaced with rational scientific theories.

  13. Enzymes with activity toward Xyloglucan

    NARCIS (Netherlands)

    Vincken, J.P.

    2003-01-01

    Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class. Here the structural variations of xyloglucans will be reviewed. Subsequently, the anchoring of xyloglucan in the plant cell wall will be discussed. Enzymes involved in degradation or modification of xyloglucan

  14. Diversity of the Germination Apparatus in Clostridium botulinum Groups I, II, III and IV

    Directory of Open Access Journals (Sweden)

    Jason Brunt

    2016-10-01

    Full Text Available Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favourable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified (encoded by various combinations of gerA, gerB and gerC genes (gerX. There are three gene clusters with an ABC-like configuration; ABC gerX1, ABABCB gerX2 and ACxBBB gerX4, and a single CA-B gerX3 gene cluster. Subtypes have been identified for most germinant receptors types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2 and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in DPA release. The cortex lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.

  15. Activation of PI3K/AKT and ERK MAPK signal pathways is required for the induction of lytic cycle replication of Kaposi's Sarcoma-associated herpesvirus by herpes simplex virus type 1

    Directory of Open Access Journals (Sweden)

    Lv Zhigang

    2011-10-01

    Full Text Available Abstract Background Kaposi's sarcoma-associated herpesvirus (KSHV is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposi's sarcoma (KS, primary effusion lymphoma (PEL and a subset of multicentric Castleman's disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1 was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV. Results By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1/signal transducer and activator of transcription 3 (STAT3 or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K/protein kinase B (PKB, also called AKT pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN and glycogen synthase kinase-3β (GSK-3β. PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK mitogen-activated protein kinase (MAPK pathway also partially contributed to HSV-1-induced KSHV replication. Conclusions HSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection.

  16. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  17. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    Science.gov (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics

    OpenAIRE

    Peng, Lixin; Chen, De; Setlow, Peter; Li, Yong-qing

    2009-01-01

    Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade spores’ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first h...

  19. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.

    2014-01-01

    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  20. Study of DNA reconstruction enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Sekiguchi, M [Kyushu Univ., Fukuoka (Japan). Faculty of Science

    1976-12-01

    Description was made of the characteristics and mechanism of 3 reconstructive enzymes which received from M. luteus or E. coli or T4, and of which natures were clarified as reconstructive enzymes of DNA irradiated with ultraviolet rays. As characteristics, the site of breaking, reaction, molecular weight, electric charge in the neutrality and a specific adhesion to DNA irradiated with ultraviolet rays were mentioned. As to mutant of ultraviolet ray sensitivity, hereditary control mechanism of removal and reconstruction by endo-nuclease activation was described, and suggestion was referred to removal and reconstruction of cells of xedoderma pigmentosum which is a hereditary disease of human. Description was also made as to the mechanism of exonuclease activation which separates dimer selectively from irradiated DNA.

  1. Metrological aspects of enzyme production

    International Nuclear Information System (INIS)

    Kerber, T M; Pereira-Meirelles, F V; Dellamora-Ortiz, G M

    2010-01-01

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies

  2. Consumer attitudes to enzymes in food production

    DEFF Research Database (Denmark)

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim

    2005-01-01

    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...... to technological progress are the socio-political attitudes that have the highest predictive value regarding attitudes to enzyme production methods....

  3. Research progress of nanoparticles as enzyme mimetics

    Science.gov (United States)

    Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun

    2011-10-01

    Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

  4. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Silica-Immobilized Enzyme Reactors

    Science.gov (United States)

    2007-08-01

    Silica-IMERs 14 implicated in neurological disorders such as Schizophrenia and Parkinson’s disease.[86] Drug discovery for targets that can alter the...primarily the activation of prodrugs and proantibiotics for cancer treatments or antibiotic therapy , respectively.[87] Nitrobenzene nitroreductase was...BuChE) Monolith disks* Packed Silica Biosilica Epoxide- Silica Silica-gel Enzyme Human AChE Human AChE Human AChE Equine BuChE Human

  6. Immobilised enzymes in biorenewable production

    OpenAIRE

    Franssen, M.C.R.; Steunenberg, P.; Scott, E.L.; Zuilhof, H.; Sanders, J.P.M.

    2013-01-01

    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, ...

  7. Immobilization of enzymes by radiation

    International Nuclear Information System (INIS)

    Kaetsu, I.; Kumakura, M.; Yoshida, M.; Asano, M.; Himei, M.; Tamura, M.; Hayashi, K.

    1979-01-01

    Immobilization of various enzymes was performed by radiation-induced polymerization of glass-forming monomers at low temperatures. Alpha-amylase and glucoamylase were effectively immobilized in hydrophilic polymer carrier such as poly(2-hydroxyethyl methacrylate) and also in rather hydrophobic carrier such as poly(tetraethylene-glycol diacrylate). Immobilized human hemoglobin underwent the reversible oxygenation concomitantly with change of oxygen concentration outside of the matrices. (author)

  8. The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

    Science.gov (United States)

    Ryon, J J; Fixman, E D; Houchens, C; Zong, J; Lieberman, P M; Chang, Y N; Hayward, G S; Hayward, S D

    1993-01-01

    Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid. Images PMID:8389916

  9. Lignin-degrading enzyme activities.

    Science.gov (United States)

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

    2012-01-01

    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

  10. Immobilised enzymes in biorenewables production.

    Science.gov (United States)

    Franssen, Maurice C R; Steunenberg, Peter; Scott, Elinor L; Zuilhof, Han; Sanders, Johan P M

    2013-08-07

    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.

  11. Self-powered enzyme micropumps

    Science.gov (United States)

    Sengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Córdova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, Ayusman

    2014-05-01

    Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

  12. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  13. The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

    Science.gov (United States)

    Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

    2017-10-01

    lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. Copyright © 2017 American Society for Microbiology.

  14. Electro-ultrafiltration of industrial enzyme solutions

    DEFF Research Database (Denmark)

    Enevoldsen, Ann Dorrit; Hansen, Erik Børresen; Jonsson, Gunnar Eigil

    2007-01-01

    To reduce the problems with fouling and concentration polarization during crossflow ultrafiltration of industrial enzyme solutions an electric field is applied across the membrane. The filtration performance during electro-ultrafiltration (EUF) has been tested with several enzymes. Results show...

  15. Biochemical characterization of thermostable cellulase enzyme from ...

    African Journals Online (AJOL)

    user

    2012-05-29

    May 29, 2012 ... tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well ... In the current industrial processes, cellulolytic enzymes ... energy sources such as glucose, ethanol, hydrogen and.

  16. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    dimer over a wide range of H+ concentrations accounts for the epigenetics of dominance for enzyme activity. [Trehan K S ... The present study has been carried on acid phosphatase .... enzyme activity over mid parent value (table 3, col. 13),.

  17. Castor Oil Transesterification Catalysed by Liquid Enzymes

    DEFF Research Database (Denmark)

    Andrade, Thalles; Errico, Massimiliano; Christensen, Knud Villy

    2017-01-01

    In the present work, biodiesel production by reaction of non-edible castor oil with methanol under enzymatic catalysis is investigated. Two liquid enzymes were tested: Eversa Transform and Resinase HT. Reactions were performed at 35 °C and with a molar ratio of methanol to oil of 6:1. The reaction...... time was 8 hours. Stepwise addition of methanol was necessary to avoid enzyme inhibition by methanol. In order to minimize the enzyme costs, the influence of enzyme activity loss during reuse of both enzymes was evaluated under two distinct conditions. In the former, the enzymes were recovered...... and fully reused; in the latter, a mixture of 50 % reused and 50 % fresh enzymes was tested. In the case of total reuse after three cycles, both enzymes achieved only low conversions. The biodiesel content in the oil-phase using Eversa Transform was 94.21 % for the first cycle, 68.39 % in the second, and 33...

  18. The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.

    Science.gov (United States)

    Almuhayawi, Mohammed; Altun, Osman; Abdulmajeed, Adam Dilshad; Ullberg, Måns; Özenci, Volkan

    2015-01-01

    Detection and identification of anaerobic bacteria in blood cultures (BC) is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux), BACTEC-Plus and -Lytic (Becton Dickinson BioSciences) BC bottles in detection and time to detection (TTD) of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94%) than BacT/ALERT FN Plus (80/100, 80%) (panaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD) and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (panaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.

  19. Zymography methods for visualizing hydrolytic enzymes

    OpenAIRE

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain

    2013-01-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

  20. Biomedical Applications of Enzymes From Marine Actinobacteria.

    Science.gov (United States)

    Kamala, K; Sivaperumal, P

    Marine microbial enzyme technologies have progressed significantly in the last few decades for different applications. Among the various microorganisms, marine actinobacterial enzymes have significant active properties, which could allow them to be biocatalysts with tremendous bioactive metabolites. Moreover, marine actinobacteria have been considered as biofactories, since their enzymes fulfill biomedical and industrial needs. In this chapter, the marine actinobacteria and their enzymes' uses in biological activities and biomedical applications are described. © 2017 Elsevier Inc. All rights reserved.

  1. Cellulolytic enzyme compositions and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Prashant; Gaspar, Armindo Ribiero; Croonenberghs, James; Binder, Thomas P.

    2017-07-25

    The present invention relates enzyme composition comprising a cellulolytic preparation and an acetylxylan esterase (AXE); and the used of cellulolytic enzyme compositions for hydrolyzing acetylated cellulosic material. Finally the invention also relates to processes of producing fermentation products from acetylated cellulosic materials using a cellulolytic enzyme composition of the invention.

  2. Immobilization of Enzymes in Polymer Supports.

    Science.gov (United States)

    Conlon, Hugh D.; Walt, David R.

    1986-01-01

    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  3. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    In the present study, the amylase enzyme producing potential of four different Aspergillus species was analyzed. The extracted amylase enzyme was purified by diethyl amino ethyl (DEAE) cellulose and Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch.

  4. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  5. PROCESS FOR DUST-FREE ENZYME MANUFACTURE

    NARCIS (Netherlands)

    Andela, C.; Feijen, Jan; Dillissen, Marc

    1994-01-01

    New enzyme granules are provided with improved properties. The granules are based on core particles having a good pore size and pore size distribution to allow an enzyme solution to enter into the particle. Accordingly, the core material comprises the enzyme in liquid form, thus eliminating the

  6. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  7. Applications of Microbial Enzymes in Food Industry

    Directory of Open Access Journals (Sweden)

    Binod Parameswaran

    2018-01-01

    Full Text Available The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

  8. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  9. Ethanologenic Enzymes of Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, Lonnie O' Neal

    1999-03-01

    Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.

  10. Prediction of Wild-type Enzyme Characteristics

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus

    of biotechnology, including enzyme discovery and characterization. This work presents two articles on sequence-based discovery and functional annotation of enzymes in environmental samples, and two articles on analysis and prediction of enzyme thermostability and cofactor requirements. The first article presents...... a sequence-based approach to discovery of proteolytic enzymes in metagenomes obtained from the Polar oceans. We show that microorganisms living in these extreme environments of constant low temperature harbour genes encoding novel proteolytic enzymes with potential industrial relevance. The second article...... presents a web server for the processing and annotation of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The third article presents analyses of the molecular determinants of enzyme thermostability, and a feature-based prediction method of the melting...

  11. Toward mechanistic classification of enzyme functions.

    Science.gov (United States)

    Almonacid, Daniel E; Babbitt, Patricia C

    2011-06-01

    Classification of enzyme function should be quantitative, computationally accessible, and informed by sequences and structures to enable use of genomic information for functional inference and other applications. Large-scale studies have established that divergently evolved enzymes share conserved elements of structure and common mechanistic steps and that convergently evolved enzymes often converge to similar mechanisms too, suggesting that reaction mechanisms could be used to develop finer-grained functional descriptions than provided by the Enzyme Commission (EC) system currently in use. Here we describe how evolution informs these structure-function mappings and review the databases that store mechanisms of enzyme reactions along with recent developments to measure ligand and mechanistic similarities. Together, these provide a foundation for new classifications of enzyme function. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?

    Science.gov (United States)

    Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin

    2017-11-01

    Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Production of Enzymes from Marine Actinobacteria.

    Science.gov (United States)

    Zhao, X Q; Xu, X N; Chen, L Y

    Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

  14. Evaluation of pressure tuning of enzymes

    DEFF Research Database (Denmark)

    Naghshineh, Mahsa

    and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer...... yield of enzymatic extraction limits the application of enzyme in pectin production. There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme...... (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste. It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE...

  15. Enzyme Enzyme activities in relation to sugar accumulation in tomato

    International Nuclear Information System (INIS)

    Alam, M.J.; Rahman, M.H.; Mamun, M.A.; Islam, K.

    2006-01-01

    Enzyme activities in tomato juice of five different varieties viz. Ratan, Marglove, BARI-1, BARI-5 and BARI-6, in relation to sugar accumulation were investigated at different maturity stages. The highest amount of invertase and beta-galactosidase was found in Marglove and the lowest in BARI- 6 at all maturity stages. Total soluble sugar and sucrose contents were highest in BARI-1 and lowest in BARI-6. The activity of amylase was maximum in Ratan and minimum in Marglove. Protease activity was highest in Ratan and lowest in BARI-6. BARI-1 contained the highest cellulase activity and the lowest in BARI-5. The amount of total soluble sugar and sucrose increased moderately from premature to ripe stage. The activities of amylase and cellulase increased up to the mature stage and then decreased drastically in the ripe stage. The activities of invertase and protease increased sharply from the premature to the ripe stage while the beta-galactosidase activity decreased remarkably. No detectable amount of reducing sugar was present in the premature stage in all cultivars of tomato but increased thereafter upto the ripe stage. The highest reducing sugar was present in BARI-5 in all of the maturity stages. (author)

  16. ENZYME RESISTANCE OF GENETICALLY MODIFIED STARCH POTATOES

    Directory of Open Access Journals (Sweden)

    A. Sh. Mannapova

    2015-01-01

    Full Text Available Here in this article the justification of expediency of enzyme resistant starch use in therapeutic food products is presented . Enzyme resistant starch is capable to resist to enzymatic hydrolysis in a small intestine of a person, has a low glycemic index, leads to decrease of postprandial concentration of glucose, cholesterol, triglycerides in blood and insulin reaction, to improvement of sensitivity of all organism to insulin, to increase in sense of fulness and to reduction of adjournment of fats. Resistant starch makes bifidogenшс impact on microflora of a intestine of the person, leads to increase of a quantity of lactobacillus and bifidobacterium and to increased production of butyric acid in a large intestine. In this regard the enzyme resistant starch is an important component in food for prevention and curing of human diseases such as diabetes, obesity, colitis, a cancer of large and direct intestine. One method is specified by authors for imitation of starch digestion in a human body. This method is based on the definition of an enzyme resistance of starch in vitro by its hydrolysis to glucose with application of a glucoamylase and digestive enzyme preparation Pancreatin. This method is used in researches of an enzyme resistance of starch, of genetically modified potato, high amylose corn starch Hi-Maize 1043 and HYLON VII (National Starch Food Innovation, USA, amylopectin and amylose. It is shown that the enzyme resistance of the starch emitted from genetically modified potatoes conforms to the enzyme resistance of the high amylose corn starch “Hi-Maize 1043 and HYLON VII starch”, (National Starch Food Innovation, the USA relating to the II type of enzyme resistant starch. It is established that amylopectin doesn't have the enzyme resistant properties. The results of researches are presented. They allow us to make the following conclusion: amylose in comparison with amylopectin possesses higher enzyme resistance and gives to

  17. [Advances on enzymes and enzyme inhibitors research based on microfluidic devices].

    Science.gov (United States)

    Hou, Feng-Hua; Ye, Jian-Qing; Chen, Zuan-Guang; Cheng, Zhi-Yi

    2010-06-01

    With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

  18. Zymography methods for visualizing hydrolytic enzymes.

    Science.gov (United States)

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E; Opdenakker, Ghislain

    2013-03-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

  19. Detoxification enzymes activities in deltamethrin and bendiocarb ...

    African Journals Online (AJOL)

    Detoxification enzymes activities in deltamethrin and bendiocarb resistant and susceptible malarial vectors ( Anopheles gambiae ) breeding in Bichi agricultural and residential sites, Kano state, Nigeria.

  20. Escherichia coli photoreactivating enzyme: purification and properties

    International Nuclear Information System (INIS)

    Snapka, R.M.; Sutherland, B.M.

    1980-01-01

    Researchers have purified large quantities of Escherichia coli photoreactivating enzyme to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36,800 and a S/sub 20,w/ 0 of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA containing uracil, adenine, guanine, and cytosine with no unusual bases detected

  1. Thermometric enzyme linked immunosorbent assay: TELISA.

    Science.gov (United States)

    Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

    1977-08-11

    A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

  2. The mechanisms of Excited states in enzymes

    DEFF Research Database (Denmark)

    Petersen, Frederic Nicolas Rønne; Bohr, Henrik

    2010-01-01

    Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes.......Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes....

  3. Genomic, proteomic and morphological characterization of two novel broad host lytic bacteriophages ΦPD10.3 and ΦPD23.1 infecting pectinolytic Pectobacterium spp. and Dickeya spp.

    Directory of Open Access Journals (Sweden)

    Robert Czajkowski

    Full Text Available Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc, P. wasabiae (Pwa and Dickeya solani (Dso with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS analysis. Pulsed-field gel electrophoresis (PFGE, genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.

  4. Spectroscopic studies of copper enzymes

    International Nuclear Information System (INIS)

    Dooley, D.M.; Moog, R.; Zumft, W.; Koenig, S.H.; Scott, R.A.; Cote, C.E.; McGuirl, M.

    1986-01-01

    Several spectroscopic methods, including absorption, circular dichroism (CD), magnetic CD (MCD), X-ray absorption, resonance Raman, EPR, NMR, and quasi-elastic light-scattering spectroscopy, have been used to probe the structures of copper-containing amine oxidases, nitrite reductase, and nitrous oxide reductase. The basic goals are to determine the copper site structure, electronic properties, and to generate structure-reactivity correlations. Collectively, the results on the amine oxidases permit a detailed model for the Cu(II) sites in these enzymes to be constructed that, in turn, rationalizes the ligand-binding chemistry. Resonance Raman spectra of the phenylhydrazine and 2,4-dinitrophenyl-hydrazine derivatives of bovine plasma amine oxidase and models for its organic cofactor, e.g. pyridoxal, methoxatin, are most consistent with methoxatin being the intrinsic cofactor. The structure of the Cu(I) forms of the amine oxidases have been investigated by X-ray absorption spectroscopy (XAS); the copper coordination geometry is significantly different in the oxidized and reduced forms. Some anomalous properties of the amine oxidases in solution are explicable in terms of their reversible aggregation, which the authors have characterized via light scattering. Nitrite and nitrous oxide reductases display several novel spectral properties. The data suggest that new types of copper sites are present

  5. Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics

    Science.gov (United States)

    Peng, Lixin; Chen, De; Setlow, Peter; Li, Yong-qing

    2009-01-01

    Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade spores’ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased ∼15% and in the second phase ending at Trelease, remaining CaDPA was released rapidly; 2) in L-alanine germination of wild-type spores and spores lacking SleB: a) the ESLI rose ∼2-fold shortly before Tlag at T1; b) following Tlag, the ESLI again rose ∼2-fold at T2 when CaDPA levels had decreased ∼50%; and c) the ESLI reached its maximum value at ∼Trelease and then decreased; 3) in CaDPA germination of wild-type spores: a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and L-alanine germination; b) at Trelease the ESLI again reached its maximum value; 4) in L-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time ΔTrelease (Trelease–Tlag) for excretion of ≥75% of CaDPA was ∼15-fold higher than that for wild-type or sleB spores; and 5) spores lacking only CwlJ exhibited a similar, but not identical ESLI pattern during L-alanine germination to that seen with cwlJ sleB spores, and the high value for ΔTrelease. PMID:19374431

  6. Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

    Science.gov (United States)

    Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

    2016-02-24

    Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  8. The use of enzymes for beer brewing

    NARCIS (Netherlands)

    Donkelaar, van Laura H.G.; Mostert, Joost; Zisopoulos, Filippos K.; Boom, Remko M.; Goot, van der Atze Jan

    2016-01-01

    The exergetic performance of beer produced by the conventional malting and brewing process is compared with that of beer produced using an enzyme-assisted process. The aim is to estimate if the use of an exogenous enzyme formulation reduces the environmental impact of the overall brewing process.

  9. Lignocellulose biotechnology: issues of bioconversion and enzyme ...

    African Journals Online (AJOL)

    Lignocellulose biotechnology: issues of bioconversion and enzyme production. ... and secondly to highlight some of the modern approaches which potentially could be used to tackle one of the major impediments, namely high enzyme cost, to speed-up the extensive commercialisation of the lignocellulose bioprocessing.

  10. Illustrating Enzyme Inhibition Using Gibbs Energy Profiles

    Science.gov (United States)

    Bearne, Stephen L.

    2012-01-01

    Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of…

  11. Enzyme Catalysis and the Gibbs Energy

    Science.gov (United States)

    Ault, Addison

    2009-01-01

    Gibbs-energy profiles are often introduced during the first semester of organic chemistry, but are less often presented in connection with enzyme-catalyzed reactions. In this article I show how the Gibbs-energy profile corresponds to the characteristic kinetics of a simple enzyme-catalyzed reaction. (Contains 1 figure and 1 note.)

  12. Enzyme Engineering for In Situ Immobilization.

    Science.gov (United States)

    Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

    2016-10-14

    Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

  13. Utilization of enzyme supplemented Telfairia occidentalis stalk ...

    African Journals Online (AJOL)

    An eight (8) week feeding trial was carried out to assess the use of enzyme natuzyme supplemented Telfairia occidentalis stalk extract as growth inducer in the practical diet for Oreochromis niloticus fingerlings. Five isonitrogenous (35% crude protein) diets at 0 ml of stalk extract and enzyme (TRT 1), 15 ml (TRT 2) and 30 ...

  14. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  15. Application of radiopolymerization for immobilization of enzymes

    International Nuclear Information System (INIS)

    Higa, O.Z.; Mastro, N.L. del; Castagnet, A.C.G.

    1986-01-01

    Hydrophilic glass-forming monomers were used in an application of irradiation technology for the immobilization of cellulase and cellobiase. Experiments to observe the effect of additives such as silicates and polyethylene glycol in the enzyme entrapment are reported on. In all cases, enzymatic activity was maintained for more than fifteen batch enzyme reactions. (Author) [pt

  16. Enzyme-Catalyzed Transetherification of Alkoxysilanes

    Directory of Open Access Journals (Sweden)

    Peter G. Taylor

    2013-01-01

    Full Text Available We report the first evidence of an enzyme-catalyzed transetherification of model alkoxysilanes. During an extensive enzymatic screening in the search for new biocatalysts for silicon-oxygen bond formation, we found that certain enzymes promoted the transetherification of alkoxysilanes when tert-butanol or 1-octanol were used as the reaction solvents.

  17. Enzymes from Higher Eukaryotes for Industrial Biocatalysis

    Directory of Open Access Journals (Sweden)

    Zhibin Liu

    2004-01-01

    Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

  18. Biocatalytic material comprising multilayer enzyme coated fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  19. 21 CFR 864.4400 - Enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme...

  20. Loop 7 of E2 enzymes

    DEFF Research Database (Denmark)

    Papaleo, Elena; Casiraghi, Nicola; Arrigoni, Alberto

    2012-01-01

    The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3...

  1. Enzyme adsorption at solid-liquid interfaces

    NARCIS (Netherlands)

    Duinhoven, S.

    1992-01-01

    Enzymes are proteins with the capacity of catalysing various reactions. Nowadays two types of enzymes, proteases and lipases, are available for use in detergent formulations for household and industrial laundry washing. Proteases are capable of catalysing the hydrolysis of proteins while

  2. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  3. Enzymic oxidation of carbon monoxide. II

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, T

    1959-01-01

    An enzyme which catalyzes the oxidation of carbon monoxide into carbon dioxide was obtained in a cell free state from Desulfovibrio desulfuricans. The enzyme activity was assayed manometrically by measuring the rate of gas uptake under the atmosphere of carbon monoxide in the presence of benzyl-viologen as an oxidant. The optimum pH range was 7 to 8. The activity was slightly suppressed by illumination. The enzyme was more stable than hydrogenase or formate dehydrogenase against the heat treatment, suggesting that it is a different entity from these enzymes. In the absence of an added oxidant, the enzyme preparation produced hydrogen gas under the atmosphere of carbon monoxide. The phenomenon can be explained assuming the reductive decomposition of water. 17 references, 4 figures, 2 tables.

  4. Enzymes - important players in green chemistry

    Directory of Open Access Journals (Sweden)

    Agata Tarczykowska

    2017-09-01

    Full Text Available Green chemistry has become a worldwide approach that leads to sustainable growth through application and development of its principles. A lot of work has to be put into designing new processes comprising of materials which do not emit pollutants to the atmosphere. Inventing new safer methods and finding less harmful products can be challenging. Enzymes are a great hope of scientists in the field of green chemistry. Enzymes as catalysts require mild conditions therefore it is a great way of saving resources such as energy or water. Processes with the use of enzymes have become more feasible by being more cost effective and eco friendly. Taking into account the benefits of green chemistry, enzyme biocatalysis has quickly replaced traditional chemical processes in several fields, and this substitution is going to reach even more areas because of new emerging technologies in enzyme engineering.

  5. Practical steady-state enzyme kinetics.

    Science.gov (United States)

    Lorsch, Jon R

    2014-01-01

    Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

  6. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

  7. Enhanced Oil Recovery with Application of Enzymes

    DEFF Research Database (Denmark)

    Khusainova, Alsu

    Enzymes have recently been reported as effective enhanced oil recovery (EOR) agents. Both laboratory and field tests demonstrated significant increase in the ultimate oil production. Up to16% of additional oil was produced in the laboratory conditions and up to 269 barrels of additional oil per day...... were recovered in the field applications. The following mechanisms were claimed to be responsible for the enhancement of the oil production due to enzymes: wettability improvement of the rock surface; formation of the emulsions; reduction of oil viscosity; and removal of high molecular weight paraffins....... However, the positive effect of enzymes on oil recovery is not that obvious. In most of the studies commercial enzyme products composed of enzymes, surfactants and stabilisers were used. Application of such samples makes it difficult to assign a positive EOR effect to a certain compound, as several...

  8. Fungal enzymes in the attine ant symbiosis

    DEFF Research Database (Denmark)

    de Fine Licht, Henrik Hjarvard; Schiøtt, Morten; Boomsma, Jacobus Jan

    the more basal attine genera use substrates such as flowers, plant debris, small twigs, insect feces and insect carcasses. This diverse array of fungal substrates across the attine lineage implies that the symbiotic fungus needs different enzymes to break down the plant material that the ants provide...... or different efficiencies of enzyme function. Fungal enzymes that degrade plant cell walls may have functionally co-evolved with the ants in this scenario. We explore this hypothesis with direct measurements of enzyme activity in fungus gardens in 12 species across 8 genera spanning the entire phylogeny...... and diversity of life-styles within the attine clade. We find significant differences in enzyme activity between different genera and life-styles of the ants. How these findings relate to attine ant coevolution and crop optimization are discussed....

  9. Production of cellulolytic enzymes from ascomycetes

    DEFF Research Database (Denmark)

    Hansen, Gustav Hammerich; Lübeck, Mette; Frisvad, Jens Christian

    2015-01-01

    Optimizing production of cellulose degrading enzymes is of great interest in order to increase the feasibility of constructing biorefinery facilities for a sustainable supply of energy and chemical products. The ascomycete phylum has a large potential for the production of cellulolytic enzymes....... Although numerous enzymatic profiles have already been unraveled, the research has been covering only a limited number of species and genera, thus leaving many ascomycetes to be analyzed. Such analysis requires choosing appropriate media and cultivation methods that ensure enzyme profiles with high...... specificities and activities. However, the choice of media, cultivation methods and enzyme assays highly affect the enzyme activity profile observed. This review provides an overview of enzymatic profiles for several ascomycetes covering phylogenetically distinct genera and species. The profiles of cellulose...

  10. Enzymes of industrial purpose - review of the market of enzyme preparations and prospects for its development

    Directory of Open Access Journals (Sweden)

    A. A. Tolkacheva

    2017-01-01

    Full Text Available Microbial enzyme preparations are increasingly replacing conventional chemical catalysts in a number of industrial processes. Such drugs, in addition to environmental friendliness and high activity, have a number of advantages over enzyme preparations of vegetable and animal origin, namely: the production of microbial enzymes in bioreactors is easily controlled and predictable; excreted microbiological enzymes are more stable than intracellular animals and plant enzymes; the genetic diversity of microorganisms makes it possible to produce enzyme preparations with a wide range of specificity; microbiological enzymes can be synthesized year-round, in contrast to the production of plant enzymes, which is often seasonal. The leaders of the world market of enzymes are proteases and amylases, which account for 25% and 15%, respectively. Over the past five years, the world market for carbohydrases, including mainly amylases, cellulases and xylanases, has been the fastest growing segment of the enzyme market with an aggregate annual growth rate of more than 7.0%. Another major product of the industrial enzyme market, which has a great potential for growth, is lipases. From the point of view of designation, the main part is represented by food and food enzymes. The Russian market continues to be unsaturated - the current supply is not able to meet the needs of the Russian feed and food industry in enzyme preparations. Enzyme preparations of domestic producers are in demand in forage production, while food industrial enterprises prefer imported products. The most significant enterprises in the enzymatic industry in Russia at the moment are Sibbiofarm, AgroSistema, Agroferment. In the light of the Russian policy of increasing food security, the development of the domestic enzyme industry is an extremely topical task.

  11. Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

    Science.gov (United States)

    Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

    2014-12-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    Science.gov (United States)

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  13. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  14. Expression of lignocellulolytic enzymes in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mellitzer Andrea

    2012-05-01

    Full Text Available Abstract Background Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic proteins due to several advantages. Results In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.

  15. Immobilized enzyme studies in a microscale bioreactor.

    Science.gov (United States)

    Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B

    2004-01-01

    Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

  16. Enzyme-based antifouling coatings: a review

    DEFF Research Database (Denmark)

    Olsen, Stefan Møller; Pedersen, Leif Toudal; Laursen, M.H.

    2007-01-01

    A systematic overview is presented of the literature that reports the antifouling (AF) protection of underwater structures via the action of enzymes. The overall aim of this review is to assess the state of the art of enzymatic AF technology, and to highlight the obstacles that have to be overcome...... for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers...

  17. Enzymic hydrolysis of cellulosic wastes to glucose

    Energy Technology Data Exchange (ETDEWEB)

    Spano, L A; Medeiros, J; Mandels, M

    1976-01-01

    An enzymic process for the conversion of cellulose to glucose is based on the use of a specific enzyme derived from mutant strains of the fungus trichoderma viride which is capable of reacting with the crystalline fraction of the cellulose molecule. The production and mode of action of the cellulase complex produced during the growth of trichoderma viride is discussed as well as the application of such enzymes for the conversion of cellulosic wastes to crude glucose syrup for use in production of chemical feedstocks, single-cell proteins, fuels, solvents, etc.

  18. Dibromine radical anion reactions with heme enzymes

    International Nuclear Information System (INIS)

    Gebicka, L.; Gebicki, J.L.

    1996-01-01

    Reactions of Br 2 radical anion with heme enzymes, catalase horseradish peroxidase, have been studied by pulse radiolysis. It has been found that Br 2 - does not react with the heme centre of investigated enzymes. Dibromine radical anion reacts with tryptophan residues of catalase without any influence on the activity of catalase. It is suggested that in pulse radiolysis studies, where horseradish peroxidase is at about tenfold excess toward Br 2 - , the enzyme is modified rather by Br 2 , than by Br 2 - . (author). 26 refs., 3 figs

  19. Dimeric assembly of enterocyte brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1994-01-01

    The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3...... appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied...

  20. Process for preparing multilayer enzyme coating on a fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  1. Dietary modulation of thymic enzymes.

    Science.gov (United States)

    Susana, Feliu María; Paula, Perris; Slobodianik, Nora

    2014-01-01

    Malnutrition is a complex syndrome caused by an inadequate intake of energy, protein, minerals and vitamins which affects the immune system. Nutritional imbalances, present in children with energy-protein malnutrition and infections, make defining the specific effects of each of them on the thymus difficult. For this reason, it is necessary to design an experimental model in animals that could define a single variable. As the thymus atrophy described in humans is similar to that observed in murines, a rat experimental model makes the extrapolation to man possible. Some authors suggest that the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP)--involved in purine metabolism--have an influence on T lymphocyte development and the immune system, due to intracellular accumulation of toxic levels of deoxynucleotides. Studies in our group, performed in an experimental model on Wistar growing rats, have demonstrated that protein deficiency or imbalance in the profile of essential amino acids in the diet, produce loss of thymus weight, reduction in the number of thymocytes, a diminished proportion of T cells presenting the W3/13 antigenic determinant and DNA content with concomitant increase in cell size, and the proportion of immature T cells and activity of ADA and PNP, without modifying the activity of 5´Nucleotidase in the thymus. It is important to point out that there were neither differences in energy intake between experimental groups and their controls, nor clinical symptoms of deficiency of other nutrients. The increase in these thymic enzyme activities was an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. On the other hand, the administration of a recovery diet, with a high amount of high quality protein, was able to reverse the mentioned effects. The quick reply of Adenosine Deaminase to nutritional disorders and the following nutritional recovery, points

  2. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    Science.gov (United States)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  3. Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

    Science.gov (United States)

    Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

    2015-03-01

    Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

  4. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    ChithrashreeGS

    2012-08-23

    Aug 23, 2012 ... peroxidase are two important antioxidant scavenging enzymes involved in ... Catalase was assayed using the method of Beers and Sizer. (1951) with .... yeast dextrose calcium carbonate agar (YDC) medium. Catalase and ...

  5. Involvement of methyltransferases enzymes during the energy

    African Journals Online (AJOL)

    Mgina

    INVOLVEMENT OF METHYLTRANSFERASES ENZYMES DURING THE. ENERGY METABOLISM OF ..... cell extract still exhibited relatively high methanogenesis with methanol (Fig ... product CH3-CoM into methane (see Fig. 1). The HS-CoM ...

  6. Enzymes: The possibility of production and applications

    Directory of Open Access Journals (Sweden)

    Petronijević Živomir B.

    2003-01-01

    Full Text Available Enzymes are biological catalysts with increasing application in the food pharmaceutical, cosmetic, textile and chemical industry. They are also important as reagents in chemical analysis, leather fabrications and as targets for the design of new drugs. Keeping in mind the growing need to replace classical chemical processes by alternative ones, because of ever growing environmental pollution, it is important that enzyme and other biotechnological processes are economical. Therefore, price decrease and stability and enzyme preparation efficiency increase are required more and more. This paper presents a short review of methods for yield increase and the improvement of the quality of enzyme products as commercial products, as well as a review of the possibilities of their application.

  7. Optimizing culture medium for debittering constitutive enzyme ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-08-02

    Aug 2, 2010 ... enzyme naringinase production by Aspergillus oryzae. JMU316. Dong-xiao .... even though industrial applications of naringinase are becoming more and ... guidance for industry. MATERIALS AND ..... For economic reasons,.

  8. distribution, abundance and properties of restriction enzymes

    African Journals Online (AJOL)

    DNA of granule-bound starch synthase (GBSS) I and II with a view to ... properties for manipulation of the genes for production of modified starch. .... procurement, storage and handling of the ..... been made on restriction enzymes of potato,.

  9. Novel enzymes for the degradation of cellulose

    Directory of Open Access Journals (Sweden)

    Horn Svein

    2012-07-01

    Full Text Available Abstract The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.

  10. Enzymes in Poultry and Swine Nutrition

    International Development Research Centre (IDRC) Digital Library (Canada)

    Poultry production in China and the potential for using enzyme preparations .... The feed manufacturers produce about 310 × 106t of high-quality feed, saving about 30%, ...... Chickens and experimental designs used in the three experiments.

  11. Archaeal Enzymes and Applications in Industrial Biocatalysts.

    Science.gov (United States)

    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  12. Polyphenol Oxidase Enzyme and Inactivation Methods

    Directory of Open Access Journals (Sweden)

    Leman Yılmaz

    2018-03-01

    Full Text Available Polyphenol oxidase enzyme is found in vegetables and fruits, as well as in some animal organs and microorganisms. Polyphenol oxidase enzyme responsible for enzymatic browning is a group of copper proteins that catalyses the oxidation of phenolic compounds to quinones, which produce brown pigments, commonly found in fruits and vegetables. During the industrial preparation of fruits and vegetables, results of catalytic effect of polyphenol oxidase causes enzymatic browning. Enzymatic browning impairs the appearance of products containing phenolic compounds along with undesirable colour, odor and taste formation and significant loss of nutritional value of the products. This affects the acceptability of the products by the consumers and causes economic losses. In this review, some characteristics of polyphenol oxidase enzyme in different fruits and vegetables have been reviewed and information about chemical antibrowning agents, thermal applications, irradiation applications and alternative methods such as high pressure processing, pulse electric field, supercritical carbon dioxide and ultrasound applications to inactivate this enzyme has been presented.

  13. Radioimmunoassay of polypeptide hormones and enzymes

    International Nuclear Information System (INIS)

    Felber, J.P.

    1974-01-01

    General principles of radioimmunoassay are reviewed. Detailed procedures are reviewed for the following hormones: insulin, pituitary hormones, gonadotropins, parathyroid hormone, ACTH, glucagon, gastrin, and peptide hormones. Radioimmunoassay of enzymes is also discussed. (U.S.)

  14. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

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

    African Journals Online (AJOL)

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

  16. Enzyme-driven mechanisms in biocorrosion

    OpenAIRE

    Basséguy, Régine

    2007-01-01

    Objectives (abstract of presentation): Recent works carried out in our team concerning enzymes and biocorrosion are presented at the meeting. For aerobic conditions, the direct catalysis of the reduction of oxygen on steel by enzymes or porphyrin was proved and a local electrochemical analysis technique (SVET) was developed to visualize the localization of the catalysis. On anaerobic conditions, the influence of phosphate species and other weak acids on the water reduction on steel was shown....

  17. A stochastic model of enzyme kinetics

    Science.gov (United States)

    Stefanini, Marianne; Newman, Timothy; McKane, Alan

    2003-10-01

    Enzyme kinetics is generally modeled by deterministic rate equations, and in the simplest case leads to the well-known Michaelis-Menten equation. It is plausible that stochastic effects will play an important role at low enzyme concentrations. We have addressed this by constructing a simple stochastic model which can be exactly solved in the steady-state. Throughout a wide range of parameter values Michaelis-Menten dynamics is replaced by a new and simple theoretical result.

  18. Enzyme Technology for Shipboard Waste Management

    Science.gov (United States)

    1976-12-01

    sucrose to the sweeter invert sugar by the enzyme invertase is a well established process, as is the conversion of starch to glucose by the enzyme...aspects of our health and daily lives. Recent advances in fundamental and applied enzymology indicate that we have already started in that direction. At a...Chemtech, p. 677 (Nov 1973) 11 - Bungay, H. P., "Applied Enzymology ," Worthington, Biochemical Corp., Notes for an AIChE Lecture, Washington, D. C. (Dec

  19. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  20. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay Rojas

    2016-01-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly a ims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes ef ficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/in activation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultr asonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  1. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay

    2016-06-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly aims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes efficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/inactivation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultrasonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  2. Directed evolution of enzymes using microfluidic chips

    Science.gov (United States)

    Pilát, Zdeněk.; Ježek, Jan; Šmatlo, Filip; Kaůka, Jan; Zemánek, Pavel

    2016-12-01

    Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate large variety of reactions, while ensuring appropriate catalytic activity and high selectivity. These properties make enzymes attractive biocatalysts for a wide range of industrial and biomedical applications. Over the last two decades, directed evolution of enzymes has transformed the field of protein engineering. We have devised microfluidic systems for directed evolution of haloalkane dehalogenases in emulsion droplets. In such a device, individual bacterial cells producing mutated variants of the same enzyme are encapsulated in microdroplets and supplied with a substrate. The conversion of a substrate by the enzyme produced by a single bacterium changes the pH in the droplet which is signalized by pH dependent fluorescence probe. The droplets with the highest enzymatic activity can be separated directly on the chip by dielectrophoresis and the resultant cell lineage can be used for enzyme production or for further rounds of directed evolution. This platform is applicable for fast screening of large libraries in directed evolution experiments requiring mutagenesis at multiple sites of a protein structure.

  3. Lysosomal enzyme activation in irradiated mammary tumors

    International Nuclear Information System (INIS)

    Clarke, C.; Wills, E.D.

    1976-01-01

    Lysosomal enzyme activity of C3H mouse mammary tumors was measured quantitatively by a histochemical method. Following whole-body doses of 3600 rad or less no changes were observed in the lysosomal enzyme activity for 12 hr after the irradiation, but very large increases in acid phosphatase and β-naphthylamidase activity were, however, observed 24 hr after irradiation. Significant increases in enzyme activity were detected 72 hr after a dose of 300 rad and the increases of enzyme activity were dose dependent over the range 300 to 900 rad. Testosterone (80 mg/kg) injected into mice 2 hr before irradiation (850 rad) caused a significant increase of lysosomal enzyme activity over and above that of the same dose of irradiation alone. If the tumor-bearing mice were given 95 percent oxygen/5 percent carbon dioxide to breathe for 8 min before irradiation the effect of 850 rad on lysosomal acid phosphatase was increased to 160 percent/that of the irradiation given alone. Activitation of lysosomal enzymes in mammary tumors is an important primary or secondary consequence of radiation

  4. Enzymes for Enhanced Oil Recovery (EOR)

    Energy Technology Data Exchange (ETDEWEB)

    Nasiri, Hamidreza

    2011-04-15

    Primary oil recovery by reservoir pressure depletion and secondary oil recovery by waterflooding usually result in poor displacement efficiency. As a consequence there is always some trapped oil remaining in oil reservoirs. Oil entrapment is a result of complex interactions between viscous, gravity and capillary forces. Improving recovery from hydrocarbon fields typically involves altering the relative importance of the viscous and capillary forces. The potential of many EOR methods depends on their influence on fluid/rock interactions related to wettability and fluid/fluid interactions reflected in IFT. If the method has the potential to change the interactions favorably, it may be considered for further investigation, i.e. core flooding experiment, pilot and reservoir implementation. Enzyme-proteins can be introduced as an enhanced oil recovery method to improve waterflood performance by affecting interactions at the oil-water-rock interfaces. An important part of this thesis was to investigate how selected enzymes may influence wettability and capillary forces in a crude oil-brine-rock system, and thus possibly contribute to enhanced oil recovery. To investigate further by which mechanisms selected enzyme-proteins may contribute to enhance oil recovery, groups of enzymes with different properties and catalytic functions, known to be interfacially active, were chosen to cover a wide range of possible effects. These groups include (1) Greenzyme (GZ) which is a commercial EOR enzyme and consists of enzymes and stabilizers (surfactants), (2) The Zonase group consists of two types of pure enzyme, Zonase1 and Zonase2 which are protease enzymes and whose catalytic functions are to hydrolyze (breakdown) peptide bonds, (3) The Novozyme (NZ) group consists of three types of pure enzyme, NZ2, NZ3 and NZ6 which are esterase enzymes and whose catalytic functions are to hydrolyze ester bonds, and (4) Alpha-Lactalbumin ( -La) which is an important whey protein. The effect of

  5. Computational Biochemistry-Enzyme Mechanisms Explored.

    Science.gov (United States)

    Culka, Martin; Gisdon, Florian J; Ullmann, G Matthias

    2017-01-01

    Understanding enzyme mechanisms is a major task to achieve in order to comprehend how living cells work. Recent advances in biomolecular research provide huge amount of data on enzyme kinetics and structure. The analysis of diverse experimental results and their combination into an overall picture is, however, often challenging. Microscopic details of the enzymatic processes are often anticipated based on several hints from macroscopic experimental data. Computational biochemistry aims at creation of a computational model of an enzyme in order to explain microscopic details of the catalytic process and reproduce or predict macroscopic experimental findings. Results of such computations are in part complementary to experimental data and provide an explanation of a biochemical process at the microscopic level. In order to evaluate the mechanism of an enzyme, a structural model is constructed which can be analyzed by several theoretical approaches. Several simulation methods can and should be combined to get a reliable picture of the process of interest. Furthermore, abstract models of biological systems can be constructed combining computational and experimental data. In this review, we discuss structural computational models of enzymatic systems. We first discuss various models to simulate enzyme catalysis. Furthermore, we review various approaches how to characterize the enzyme mechanism both qualitatively and quantitatively using different modeling approaches. © 2017 Elsevier Inc. All rights reserved.

  6. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  7. La dicotomía de los virus polioma: ¿Infección lítica o inducción de neoplasias? The paradox of polyomaviruses Lytic infection or tumor induction?

    Directory of Open Access Journals (Sweden)

    Norberto A. Sanjuan

    2004-02-01

    Full Text Available Los virus Polioma murinos provocan infecciones líticas en cultivos de células de ratón y transforman in vitro células de rata a través de la interacción de su oncogén mT con diversos reguladores celulares. Luego de su inoculación en ratones neonatos inducen neoplasias epiteliales y mesenquimáticas. Se ha propuesto que las cepas de polioma más oncogénicas son aquellas que previamente replican más en el ratón. Sin embargo, a nivel de una sola célula la infección lítica y la transformación deberían ser mutuamente excluyentes. En cada neoplasia han sido descriptos 3 tipos celulares según expresen el DNA viral solo o concomitantemente con la proteína mayor de la cápside VP1, o que no contengan DNA viral ni VP-1. En nuestro laboratorio detectamos la existencia de un cuarto tipo celular en las neoplasias, en el que se expresa la totalidad del genoma viral pero no ocurre el ensamblaje, probablemente por alteraciones en la fosforilación de VP-1. Se discuten los mecanismos de migración intracelular de Polioma, la diseminación en el ratón y los factores que podrían estar involucrados en la inducción de neoplasias o en la infección lítica inducidas por el virus.Murine polyomaviruses can produce lytic infections in mouse cell cultures or transform in vitro rat fibroblasts through a complex interaction with key cellular regulators. After infection of newborn mice, some strains of polyomavirus induce epithelial and mesenchymal tumors. It has been described that there is a direct relationship between viral dissemination in the mouse and tumor induction. However, at a single cell level lytic infection and transformation would not be able to coexist. The existence of 3 distinct cell populations in polyoma-induced tumors, classified according to the presence or absence of viral DNA and viral capsid protein VP-1 have been described. We have reported a fourth type of cell in the neoplasms, that can express the early and the late viral

  8. Enzyme-MOF (metal-organic framework) composites.

    Science.gov (United States)

    Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

    2017-06-06

    The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

  9. Kinetics of enzyme action: essential principles for drug hunters

    National Research Council Canada - National Science Library

    Stein, Ross L

    2011-01-01

    ... field. Beginning with the most basic principles pertaining to simple, one-substrate enzyme reactions and their inhibitors, and progressing to a thorough treatment of two-substrate enzymes, Kinetics of Enzyme Action...

  10. Continuous enzyme reactions with immobilized enzyme tubes prepared by radiation cast-polymerization

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1986-01-01

    Immobilized glucose oxidase tubes were prepared by radiation cast-polymerization of 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate monomer at low temperatures. The immobilized enzyme tubes which were spirally set in a water bath were used as reactor, in which the enzyme activity varied with tube size and flow rate of the substrate. The conversion yield of the substrate in continuous enzyme reaction was about 80%. (author)

  11. Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

    International Nuclear Information System (INIS)

    AuCoin, David P.; Colletti, Kelly S.; Cei, Sylvia A.; Papouskova, Iva; Tarrant, Margaret; Pari, Gregory S.

    2004-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

  12. Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds.

    Directory of Open Access Journals (Sweden)

    Panos G Kalatzis

    Full Text Available Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.

  13. Isolation and Characterization of a Lytic Bacteriophage (vB_PmiS-TH) and Its Application in Combination with Ampicillin against Planktonic and Biofilm Forms of Proteus mirabilis Isolated from Urinary Tract Infection.

    Science.gov (United States)

    Yazdi, Mahsa; Bouzari, Majid; Ghaemi, Ezzat Allah

    2018-01-01

    Proteus mirabilis is one of the most common causes of urinary tract infection (UTI), particularly in patients undergoing long-term catheterization. Phage vB_PmiS-TH was isolated from wastewater with high lytic activity against P. mirabilis (TH) isolated from UTI. The phage had rapid adsorption, a large burst size (∼260 PFU per infected cell), and high stability at a wide range of temperatures and pH values. As analyzed by transmission electron microscopy, phage vB_PmiS-TH had an icosahedral head of ∼87 × 62 nm with a noncontractile tail about 137 nm in length and 11 nm in width. It belongs to the family Siphoviridae. Combination of the phage vB_PmiS-TH with ampicillin had a higher removal activity against planktonic cells of P. mirabilis (TH) than the phage or the antibiotic alone. Combination of the phage at a multiplicity of infection of 100 with a high dose of ampicillin (246 µg/mL) showed the highest biofilm removal activity after 24 h. This study demonstrates that using a combination of phage and antibiotic could be significantly more effective against planktonic and biofilm forms of P. mirabilis (TH). © 2018 S. Karger AG, Basel.

  14. Combined effect of synthetic enterocin CRL35 with cell wall, membrane-acting antibiotics and muranolytic enzymes against Listeria cells.

    Science.gov (United States)

    Salvucci, E; Hebert, E M; Sesma, F; Saavedra, L

    2010-08-01

    To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane-acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.

  15. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    Science.gov (United States)

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  16. Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices

    OpenAIRE

    Munteanu, Cristian Robert

    2014-01-01

    Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices, German Conference on Bioinformatics (GCB), Potsdam, Germany (September, 2007)

  17. Enzymic conversion of starch to glucose

    Energy Technology Data Exchange (ETDEWEB)

    1964-08-19

    Corn is steeped in a SO/sub 2/ solution for 30 to 40 hours, coarsely ground, separated from the germ, and filtered. A 35% suspension of the germ-free corn, still containing fibers, hull, and gluten, is treated with Ca(OH)/sub 2/ to raise the pH to 6.5 to 7.0. A starch-liquifying enzyme is added and after a 2 hours treatment at 85/sup 0/ the liquefied starch is cooled to 60/sup 0/ and the pH is adjusted to 4.5 to 5.0 with H/sub 2/SO/sub 4/. A saccharifying enzyme is now added. After 40 to 81 hours, a raw glucose solution is obtained and is freed from fibers and gluten by filtration. The commercial starch-liquifying enzymes are designated HT-1000 and Neozyme 3 LC (liquid). The saccharifying enzymes are Diazyme or Diazyme L 30 (liquid). The solid enzymes are used at a level up to 0.1% by weight of the starch. Up to 100% conversion of starch into glucose is achieved.

  18. Thermophilic archaeal enzymes and applications in biocatalysis.

    Science.gov (United States)

    Littlechild, Jennifer A

    2011-01-01

    Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

  19. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  20. Development of enzymes and enzyme systems by genetic engineering to convert biomass to sugars

    Science.gov (United States)

    TITLE Development of Enzymes and Enzyme Systems by Genetic Engineering to Convert Biomass to Sugars ABSTRACT Plant cellulosic material is one of the most viable renewable resources for the world’s fuel and chemical feedstock needs. Currently ethanol derived from corn starch is the most common li...

  1. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  2. Microbial genetic engineering and enzyme technology

    Energy Technology Data Exchange (ETDEWEB)

    Hollenberg, C.P.; Sahm, H.

    1987-01-01

    In a series of up-to-date contributions BIOTEC 1 has experts discussing the current topics in microbial gene technology and enzyme technology and speculating on future developments. Bacterial and yeast systems for the production of interferons, growth hormone or viral antigenes are described as well as the impact of gene technology on plants. Exciting is the prospect of degrading toxic compounds in our environment by microorganisms tuned in the laboratory. Enzymes are the most effective catalysts we know. They exhibit a very high substrate- and stereospecificity. These properties make enzymes extremely attractive as industrial catalysts, leading to new production processes that are non-polluting and save both energy and raw materials. (orig.) With 135 figs., 36 tabs.

  3. Ultrasound in Enzyme Activation and Inactivation

    Science.gov (United States)

    Mawson, Raymond; Gamage, Mala; Terefe, Netsanet Shiferaw; Knoerzer, Kai

    As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.

  4. Enzyme Histochemistry for Functional Histology in Invertebrates.

    Science.gov (United States)

    Cima, Francesca

    2017-01-01

    In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.

  5. Intestinal enzyme distribution after supralethal irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Becciolini, A; Gerber, G B; Buracchi, A; Deroo, J [Florence Univ. (Italy). Istituto di Radiologia; Centre d' Etude de l' Energie Nucleaire, Mol (Belgium). Dept. de Radiobiologie)

    1977-07-01

    The activity of some intestinal enzymes has been studied after 2 kR irradiation. Brush border enzymes, maltase and leucineaminopeptidase (LAP) show an increase 20 hours after irradiation, while after 72 hours their activities are reduced to very low levels. Lysosomal enzymes show a completely different behaviour: acid phosphatase activity increases only 72 hours after irradiation, whereas ..beta.. glucuronidase increases significantly after 20 hours and reaches values two or three times higher than controls after 72 hours. The histologic picture at the first interval after irradiation shows gross alterations in the crypt region, but the villi appear nearly normal. Seventy-two hours after irradiation the whole epithelium is affected and very numerous leukocytes are present in the stroma.

  6. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  7. Radiation and enzyme degradation of cellulose materials

    International Nuclear Information System (INIS)

    Duchacek, V.

    1983-01-01

    The results are summed up of a study of the effect of gamma radiation on pure cellulose and on wheat straw. The irradiation of cellulose yields acid substances - formic acid and polyhydroxy acids, toxic malondialdehyde and the most substantial fraction - the saccharides xylose, arabinose, glucose and certain oligosaccharides. A ten-fold reduction of the level of cellulose polymerization can be caused by relatively small doses - (up to 250 kGy). A qualitative analysis was made of the straw before and after irradiation and it was shown that irradiation had no significant effect on the qualitative composition of the straw. A 48 hour enzyme hydrolysis of the cellulose and straw were made after irradiation and an economic evaluation of the process was made. Radiation pretreatment is technically and economically advantageous; the production of fodder using enzyme hydrolysis of irradiated straw is not economically feasible due to the high cost of the enzyme. (M.D.)

  8. Engineering of pectinolytic enzymes for enhanced thermostability

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller

    Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

  9. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only...... about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained...

  10. Enhanced Oil Recovery with Application of Enzymes

    OpenAIRE

    Khusainova, Alsu; Shapiro, Alexander; Woodley, John

    2016-01-01

    Enzymer er for nylig blevet rapporteret, som effektive stoffer for forbedret olieindvinding(EOR). Både laboratorie undersøgelser og felttest viste en markant stigning af olieproduktion. Op til ekstra 16 % af olien blev produceret i laboratorie eksperimenter og op til ekstra 269 tønder olie per dag blev fremstillet under feltforsøg. Det var foreslået, at følgende mekanismer har medvirket tiløget olieproduktionen på grund af enzymer: forbedringer af bjergartsoverfladens befugtningsevne;dannelse...

  11. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    International Nuclear Information System (INIS)

    Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

    2006-01-01

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

  12. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement of...

  13. 21 CFR 864.9400 - Stabilized enzyme solution.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

  14. Review of the biochemical basis of enzyme immunoassays

    International Nuclear Information System (INIS)

    Klingler, W.

    1982-01-01

    The ever increasing number of radioimmunological determination poses problems allied with the handling of radioactive substances. In recent years various non-radioactive methods have been developed, among which the enzyme immunoassay is already in routine use. Homogeneous and heterogeneous enzyme immunoassays are described. Criteria for enzymes, substrates and enzyme-substrate reactions are listed. (orig.) [de

  15. Seeing & Feeling How Enzymes Work Using Tangible Models

    Science.gov (United States)

    Lau, Kwok-chi

    2013-01-01

    This article presents a tangible model used to help students tackle some misconceptions about enzyme actions, particularly the induced-fit model, enzyme-substrate complementarity, and enzyme inhibition. The model can simulate how substrates induce a change in the shape of the active site and the role of attraction force during enzyme-substrate…

  16. The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

    Science.gov (United States)

    Gupta, Deepak K; Rühl, Martin; Mishra, Bagdevi; Kleofas, Vanessa; Hofrichter, Martin; Herzog, Robert; Pecyna, Marek J; Sharma, Rahul; Kellner, Harald; Hennicke, Florian; Thines, Marco

    2018-01-15

    Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

  17. Purification, characterization of phytase enzyme from Lactobacillus ...

    African Journals Online (AJOL)

    Purification, characterization of phytase enzyme from Lactobacillus plantarum bacteria and determination of its kinetic properties. ... Many of the cereal grains, legumes and oilseeds store phosphorus in phytate form. Phytases can be produced by plants, animals and microorganisms. However, the ones with microbial origin ...

  18. Enzyme Replacement Therapy for Fabry Disease

    Directory of Open Access Journals (Sweden)

    Maria Dolores Sanchez-Niño PhD

    2016-11-01

    Full Text Available Fabry disease is a rare X-linked disease caused by the deficiency of α-galactosidase that leads to the accumulation of abnormal glycolipid. Untreated patients develop potentially lethal complications by age 30 to 50 years. Enzyme replacement therapy is the current standard of therapy for Fabry disease. Two formulations of recombinant human α-galactosidase A (agalsidase are available in most markets: agalsidase-α and agalsidase-β, allowing a choice of therapy. However, the US Food and Drug Administration rejected the application for commercialization of agalsidase-α. The main difference between the 2 enzymes is the dose. The label dose for agalsidase-α is 0.2 mg/kg/2 weeks, while the dose for agalsidase-β is 1.0 mg/kg/2 weeks. Recent evidence suggests a dose-dependent effect of enzyme replacement therapy and agalsidase-β is 1.0 mg/kg/2 weeks, which has been shown to reduce the occurrence of hard end points (severe renal and cardiac events, stroke, and death. In addition, patients with Fabry disease who have developed tissue injury should receive coadjuvant tissue protective therapy, together with enzyme replacement therapy, to limit nonspecific progression of the tissue injury. It is likely that in the near future, additional oral drugs become available to treat Fabry disease, such as chaperones or substrate reduction therapy.

  19. Enzyme catalysis by entropy without Circe effect.

    Science.gov (United States)

    Kazemi, Masoud; Himo, Fahmi; Åqvist, Johan

    2016-03-01

    Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction in water, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

  20. Biochemical assessement of liver enzymes in immunocompromised ...

    African Journals Online (AJOL)

    Aim: This study aims at the estimation of serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and glutmyltransferase GGT (Liver enzymes) in Human immunodeficiency virus(HIV) and/or Acquired immune deficiency syndrome(AIDS) patients in parts of Edo State, Nigeria.

  1. Enzyme specific activity in functionalized nanoporous supports

    International Nuclear Information System (INIS)

    Lei Chenghong; Soares, Thereza A; Shin, Yongsoon; Liu Jun; Ackerman, Eric J

    2008-01-01

    Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P LD ) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I e , defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH 2 - and HOOC-functionalized mesoporous silica (300 A, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P LD . With decreasing P LD , I e of GOX in FMS increased from 150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P LD . With increasing P LD , the corresponding I e of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P LD , consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P LD and may promote a more favorable confinement environment that enhances the OPH activity

  2. Exogenous fibrolytic enzymes to unlock nutrients: Histological ...

    African Journals Online (AJOL)

    There is a need for a better understanding of the mode-of-action of exogenous fibrolytic enzymes (EFE) used as additives in ruminant feeds. Four forages, treated with EFE, were evaluated in vitro and histologically, in an attempt to determine the effect of EFE on tissue degradation. Weeping love grass, kikuyu leaf material, ...

  3. Microbial nitrilases: versatile, spiral forming, industrial enzymes

    CSIR Research Space (South Africa)

    Thuku, RN

    2009-03-01

    Full Text Available such case is the NAD+ synthetase from Mycobacterium tuberculosis (Bellinzoni et al., 2005). This enzyme relies on an associated amino-terminal amidase domain in order to utilize glutamine as a source of nitrogen and liberate ammonia which is required...

  4. A Comprehensive Enzyme Kinetic Exercise for Biochemistry

    Science.gov (United States)

    Barton, Janice S.

    2011-01-01

    This article describes a comprehensive treatment of experimental enzyme kinetics strongly coupled to electronic data acquisition and use of spreadsheets to organize data and perform linear and nonlinear least-squares analyses, all in a manner that promotes development of important reasoning skills. Kinetic parameters are obtained for the stable…

  5. A Qualitative Approach to Enzyme Inhibition

    Science.gov (United States)

    Waldrop, Grover L.

    2009-01-01

    Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…

  6. Enzyme Kinetics? Elementary, my dear 3 -8 ...

    Indian Academy of Sciences (India)

    research interests are in the areas of protein- ... rate constant for the formation of products, k3 is significantly of some enzymes. ... tissue at different stages of development. .... represent the only values of Km and V max that satisfy all of the sets.

  7. Angiotensin Converting Enzyme Insertion/Deletion Gene ...

    African Journals Online (AJOL)

    Angiotensin Converting Enzyme Insertion/Deletion Gene Polymorphism: An Observational Study among Diabetic Hypertensive Subjects in Malaysia. ... Methods: The pharmacological effect of ACE inhibition on mean arterial pressure (MAP) and glomerular filtration rate (GFR) were observed among a total of 62 subjects for ...

  8. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M

    2013-01-01

    opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...... concentrations, and stability. Finally, future research directions are discussed, including the integration of biocatalysis with neighboring chemical steps....

  9. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused CBHI.1 protein. Plate enzyme ...

  10. Detergents - Zeolites and Enzymes Excel Cleaning Power

    Indian Academy of Sciences (India)

    Presently used detergent formulations generally consist of surfactants, builder and cobuilder, bleaching agents, addi- tives for secondary benefits and enzymes. Zeolites are basically hydrated crystalline aluminium silicates which function as ion exchangers and make the water soft by removing calcium, magnesium and ...

  11. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    Science.gov (United States)

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  12. Furin is a chemokine-modifying enzyme

    DEFF Research Database (Denmark)

    Hensbergen, Paul J; Verzijl, Dennis; Balog, Crina I A

    2004-01-01

    Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the tota...

  13. Ligninolytic enzyme complex of Armillaria spp

    Czech Academy of Sciences Publication Activity Database

    Stoychev, I.; Nerud, František

    2000-01-01

    Roč. 45, č. 3 (2000), s. 248-250 ISSN 0015-5632 Institutional research plan: CEZ:AV0Z5020903 Keywords : lignin olytic enzyme * lignin peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 0.752, year: 2000

  14. Involvement of methyltransferases enzymes during the energy ...

    African Journals Online (AJOL)

    The methyl group transfer from dimethylsulfide (DMS), trimethylamine and methanol to 2-mercaptoethanesulfonic acid (coenzyme M) were investigated from cell extracts of Methanosarcina semesiae sp. nov. to evaluate whether the enzyme systems involved were constitutive or inductive. The extracts from cells grown on ...

  15. Computationally designed libraries for rapid enzyme stabilization

    NARCIS (Netherlands)

    Wijma, Hein J.; Floor, Robert J.; Jekel, Peter A.; Baker, David; Marrink, Siewert J.; Janssen, Dick B.

    The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants

  16. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    In the present work, we studied the role of antioxidant scavenging enzymes of plant pathogenic bacteria: catalase, ascorbate peroxidase and a virulence factor; extracelluar polysaccharide production in determining the virulence of Xanthomonas oryzae pv. oryzae (Xoo) isolates and its differential reaction to rice cultivars.

  17. Novel Industrial Enzymes from Uncultured Arctic Microorganisms

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede

    , and reduce the risk of contaminations. Cold- and alkaline-active enzymes can be found in microorganisms adapted to living in natural environments with these conditions, which are extremely rare but found in the unique ikaite columns from SW Greenland (4-6 °C, pH >10). It is estimated that less than 1...

  18. Microbial nitrilases: versatile, spiral forming, industrial enzymes.

    Science.gov (United States)

    Thuku, R N; Brady, D; Benedik, M J; Sewell, B T

    2009-03-01

    The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

  19. Enzymes and Ecosystems -- Where Do They Overlap?

    Science.gov (United States)

    Richard E. Dickson

    1996-01-01

    The whole plant is not the sum of its enzyme systems. This book demonstrates the importance of whole-plant physiology by examining carbon-nitrogen interactions and how these interactions are influenced by demands of the whole plant. In some aspects it is a timely response to the current, strong reductionist trends in plant physiology associated with advances in...

  20. Transition state theory for enzyme kinetics

    Science.gov (United States)

    Truhlar, Donald G.

    2015-01-01

    This article is an essay that discusses the concepts underlying the application of modern transition state theory to reactions in enzymes. Issues covered include the potential of mean force, the quantization of vibrations, the free energy of activation, and transmission coefficients to account for nonequilibrium effect, recrossing, and tunneling. PMID:26008760

  1. Synthetic Applications of Nitrile-Converting Enzymes

    Czech Academy of Sciences Publication Activity Database

    Martínková, Ludmila; Mylerová, Veronika

    2003-01-01

    Roč. 7, - (2003), s. 1279-1295 ISSN 1385-2728 R&D Projects: GA AV ČR IAA4020213 Institutional research plan: CEZ:AV0Z5020903 Keywords : nitrile * converting * enzymes Subject RIV: EE - Microbiology, Virology Impact factor: 2.521, year: 2003

  2. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  3. Chitinolytic enzymes produces by ovine rumen bacteria

    Czech Academy of Sciences Publication Activity Database

    Kopečný, Jan; Hodrová, Blanka

    2000-01-01

    Roč. 45, č. 5 (2000), s. 465-468 ISSN 0015-5632 R&D Projects: GA ČR GA524/97/1221 Institutional research plan: CEZ:AV0Z5045916 Keywords : chitinolytic enzymes Subject RIV: ED - Physiology Impact factor: 0.752, year: 2000

  4. growth and extracellular enzyme production by microorganisms

    African Journals Online (AJOL)

    Okorie

    2013-06-26

    Jun 26, 2013 ... 1Federal Institute of Industrial Research Oshodi, Lagos, Nigeria. 2Department of ... of Bacillus subtilis (Bs2) were able to produce lipase enzyme. The study ... However, most commercial starter cultures originated from those ... The traditional method of preparing Ugba was employed in the laboratory to ...

  5. Laccase Enzymes in Inocula Pleurotus spp

    Directory of Open Access Journals (Sweden)

    Nora García-Oduardo

    2017-01-01

    Full Text Available The cultivation of edible and medicinal mushrooms Pleurotus has been aimed at promoting alternative management for agricultural products. This basidiomicete has been the subject of numerous studies because of its fruiting body constitutes a food, being a producer of enzymes with industrial interest and for its ability of biotransformation of lignocellulosic substrates. Pleurotus inocula in the established technology for growing edible and medicinal mushrooms in the CEBI Research- Production Plant were performed using sorghum or wheat. However, it is possible to expand the possibilities with other substrates. In this paper, the results of laccase enzymes production in inocula prepared with sorghum, corn and coffee pulp with two strains Pleurotus ostreatus CCEBI 3021 and Pleurotus ostreatus CCEBI 3024 are presented. The period of preparation of seed reaches 15-21 days, the measurements of laccase activity were performed in periods of seven days. Extraction of crude enzyme was performed in aqueous phase, the determination of the laccase enzyme activity, using guaiacol as substrate. The results obtained in this work with studies in previous work using sorghum as inocula are compared. It is found that higher yields are obtained laccase in coffee pulp. This study contributes to the theoretical knowledge and to provide alternatives for securing the production process of the plant.

  6. Lignocellulose degradation, enzyme production and protein ...

    African Journals Online (AJOL)

    Microbial conversion of corn stover by white rot fungi has the potential to increase its ligninolysis and nutritional value, thereby transforming it into protein-enriched animal feed. Response surface methodology was applied to optimize conditions for the production of lignocellulolytic enzymes by Trametes versicolor during ...

  7. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wösten-van Asperen, Roelie M.; Bos, Albert P.; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, René

    2013-01-01

    Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin

  8. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wosten-van Asperen, Roelie M.; Bos, Albert; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, Rene

    2013-01-01

    Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts

  9. Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions.

    Science.gov (United States)

    Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C

    2017-01-01

    The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

  10. Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

    Science.gov (United States)

    Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

    2011-01-01

    The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

    International Nuclear Information System (INIS)

    Schachinger, L.; Schippel, C.; Altmann, E.; Diepold, B.; Yang, C.; Jaenike, M.; Hochhaeuser, E.

    1985-01-01

    Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldialdehyde according to Morris, Campell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O 2 -uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Ksub(m)-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Ksub(m)-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier. (orig.)

  12. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

    Science.gov (United States)

    Leurs, Melanie; Tiller, Joerg C

    2017-01-01

    The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

  13. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  14. Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution.

    Science.gov (United States)

    Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V

    2010-04-30

    Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.

  15. Therapeutic Enzymes: Applications and Approaches to Pharmacological Improvement.

    Science.gov (United States)

    Yari, Maryam; Ghoshoon, Mohammad B; Vakili, Bahareh; Ghasemi, Younes

    2017-01-01

    Among therapeutic proteins, enzymes represent small and of course profitable market. They can be used to treat important, rare, and deadly diseases. Enzyme therapy is the only available treatment for certain disorders. Here, pharmaceutical enzymes are reviewed. They are categorized in four main groups, enzymes in replacement therapy, enzymes in cancer treatment, enzymes for fibrinolysis, and finally enzymes that are used topically for various treatments. Furthermore, enzyme gene therapy and future perspective of therapeutic enzymes are mentioned in brief. There are many important approved enzymes in pharmaceutical market. Several approaches such as point mutation, fusion protein designing, glycoengineering, and PEGylation were used to achieve improved enzymes. Although sometimes enzymes were engineered to facilitate production and purification process, appropriate delivery to target sites, extending half-life, and reducing immunogenicity are among the main goals of engineering approaches. Overall, enzymes play a critical role in treatment of common and rare diseases. Evaluation of new enzymes as well as improvement of approved enzymes are of the most important challenges in biotechnology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

    Science.gov (United States)

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2015-12-01

    The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

    Science.gov (United States)

    Sirisha, V L; Jain, Ankita; Jain, Amita

    Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

  18. Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting

    Directory of Open Access Journals (Sweden)

    Michael J. Allen

    2011-04-01

    Full Text Available Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house‘ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

  19. Enzymic saccharification of some pretreated agricultural wastes

    Energy Technology Data Exchange (ETDEWEB)

    El-Gammal, S.M.A.; Sadek, M.A.

    1988-01-01

    Cellulosie wastes, artichoke leaves and stalks, sugar-cane bagasse and fennel seeds after extraction of essential oils were treated with various concentrations of peracetic acid at 100/sup 0/C, 60/sup 0/C and room temperature several times, washed with water and ethanol and air dried. The degree of enzymatic solubilization of each treated cellulosic waste was measured with Aspergillus niger cellulase (Endo-1,4-B-Glucanase; 1,4-(1,3; 1,4)-..beta..-D-glucan 4-glucanohydrolase; EC 3. 2.1.4). Artichoke waste and sugar-cane bagasse were solubilized more efectively by the enzymethan fennel waste. Data are presented describing the effect of time, enzyme and substrate concentration on the rate of enzymic hydrolysis. Infrared spectra of the treated and untreated cellulosic materials were recorded.

  20. Hydrolytic enzyme activity enhanced by Barium supplementation

    Directory of Open Access Journals (Sweden)

    Camilo Muñoz

    2016-10-01

    Full Text Available Hydrolysis of polymers is a first and often limiting step during the degradation of plant residues. Plant biomass is generally a major component of waste residues and a major renewable resource to obtain a variety of secondary products including biofuels. Improving the performance of enzymatic hydrolysis of plant material with minimum costs and limiting the use of additional microbial biomass or hydrolytic enzymes directly influences competitiveness of these green biotechnological processes. In this study, we cloned and expressed a cellulase and two esterases recovered from environmental thermophilic soil bacterial communities and characterize their optimum activity conditions including the effect of several metal ions. Results showed that supplementing these hydrolytic reactions with Barium increases the activity of these extracellular hydrolytic enzymes. This observation represents a simple but major improvement to enhance the efficiency and competitiveness of this process within an increasingly important biotechnological sector.

  1. (Hyper)thermophilic enzymes: production and purification.

    Science.gov (United States)

    Falcicchio, Pierpaolo; Levisson, Mark; Kengen, Servé W M; Koutsopoulos, Sotirios

    2014-01-01

    The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

  2. A thermodynamic and theoretical view for enzyme regulation.

    Science.gov (United States)

    Zhao, Qinyi

    2015-01-01

    Precise regulation is fundamental to the proper functioning of enzymes in a cell. Current opinions about this, such as allosteric regulation and dynamic contribution to enzyme regulation, are experimental models and substantially empirical. Here we proposed a theoretical and thermodynamic model of enzyme regulation. The main idea is that enzyme regulation is processed via the regulation of abundance of active conformation in the reaction buffer. The theoretical foundation, experimental evidence, and experimental criteria to test our model are discussed and reviewed. We conclude that basic principles of enzyme regulation are laws of protein thermodynamics and it can be analyzed using the concept of distribution curve of active conformations of enzymes.

  3. Ethanol from wood. Cellulase enzyme production

    Energy Technology Data Exchange (ETDEWEB)

    Szengyel, Zsolt

    2000-03-01

    Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment.

  4. Substrates and method for determining enzymes

    Science.gov (United States)

    Smith, R.E.; Bissell, E.R.

    1981-10-13

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

  5. Nedd8 processing enzymes in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    O'Donoghue, Jean; Bech-Otschir, Dawadschargal; Larsen, Ida

    2013-01-01

    Conjugation of the ubiquitin-like modifier Nedd8 to cullins is critical for the function of SCF-type ubiquitin ligases and thus facilitates ubiquitin conjugation and ultimately degradation of SCF substrates, including several cell cycle regulators. Like ubiquitin, Nedd8 is produced as a precursor...... that must first be processed before it becomes active. In Saccharomyces cerevisiae this is carried out exclusively by the enzyme Yuh1....

  6. Radioisotope-enzymes and cancer study

    International Nuclear Information System (INIS)

    Luyen, T. van

    2008-01-01

    Cancer is a pathological sign, when the abnormal cells appear in certain human tissues or organs. These cells can reproduce beyond the control of normal biological protection mechanism. Because they reproduce very fast, the metabolic process is accelerated, which causes the extreme need for more energy, substrate and catalyzing enzymes. Based on these needs, we can control the metabolic process by: Stopping supplying the energy. Stopping supplying the substrate and the materials to build up the cell's structure. Stopping operating catalysis by breaking out the enzyme's structure. Destroying the tumor cell by extra agents such as radiations and chemicals. All of these methods have been studied for a long time, which costs too much money, time and labor. Although we succeeded in some ways, the results are still not satisfactory. There are many reasons for this situation but the main one is the lack of information to understand all the processes taking place in the cell and our body. However, as far as we studied, we would like to propose the method to break the structure of the enzyme by nuclear decay process. (author)

  7. Effect of turmeric on xenobiotic metabolising enzymes.

    Science.gov (United States)

    Goud, V K; Polasa, K; Krishnaswamy, K

    1993-07-01

    Diet contains several substances capable of inhibiting chemical carcinogenesis. It is known that such inhibitors may either act directly by scavenging the reactive substances or indirectly by promoting mechanisms which enhance detoxification. Turmeric which contains curcumin both in vitro and in vivo is an active antimutagen. Studies were therefore conducted to evaluate the effects of turmeric on xenobiotic metabolising enzymes in hepatic tissue of rats fed turmeric ranging from 0.5-10% in the diet. Enzymes such as aryl hydrocarbon hydroxylase, UDP glucuronyl transferase and glutathione-S-transferase were assayed after four weeks of turmeric fed diets. No significant differences were seen in the activating enzyme AHH. However, UDPGT was significantly elevated in rats fed 10% turmeric while GSHT registered a significant increase in 5 and 10% turmeric fed diet as compared to controls and 0.5-1.0% turmeric fed animals. The results suggest that turmeric may increase detoxification systems in addition to its anti-oxidant properties. Curcumin perhaps is the active principle in turmeric. Turmeric used widely as a spice would probably mitigate the effects of several dietary carcinogens.

  8. Enzyme-Gelatin Electrochemical Biosensors: Scaling Down

    Directory of Open Access Journals (Sweden)

    Hendrik A. Heering

    2012-03-01

    Full Text Available In this article we investigate the possibility of scaling down enzyme-gelatin modified electrodes by spin coating the enzyme-gelatin layer. Special attention is given to the electrochemical behavior of the selected enzymes inside the gelatin matrix. A glassy carbon electrode was used as a substrate to immobilize, in the first instance, horse heart cytochrome c (HHC in a gelatin matrix. Both a drop dried and a spin coated layer was prepared. On scaling down, a transition from diffusion controlled reactions towards adsorption controlled reactions is observed. Compared to a drop dried electrode, a spin coated electrode showed a more stable electrochemical behavior. Next to HHC, we also incorporated catalase in a spin coated gelatin matrix immobilized on a glassy carbon electrode. By spincoating, highly uniform sub micrometer layers of biocompatible matrices can be constructed. A full electrochemical study and characterization of the modified surfaces has been carried out. It was clear that in the case of catalase, gluteraldehyde addition was needed to prevent leaking of the catalase from the gelatin matrix.

  9. Ionizing radiation effect on enzymes. III

    International Nuclear Information System (INIS)

    Libicky, A.; Chottova, O.; Fidlerova, J.; Urban, J.; Kubankova, V.

    1980-01-01

    A decrease in the efficacy of trypsin (determination according to PhBs 3 with the use of L-lysine ethyl ester chloride) was investigated in pancreatin obtained by enzyme precipitation from a pancreas extraction after autolysis, in the identical sample with an additionally increased content of lipids, in pancreatin containing parts of the pancreatic tissue, in crystalline trypsin, and in crystalline salt-free and lyophilized trypsine after irradiation with gamma rays from 60 Co, doses ranging from 1x10 4 Gy to 12x10 4 Gy. The results were statistically evaluated and after the conversion to dried or lipid-free substance expressed in graphs. The dependence of the efficacy on the radiation dose has a linear course in semi-logarithmic arrangement, similarly as it occurred in chymotrypsin and in the total proteolytic efficacy. The decrease in the efficacy of trypsin in the samples of pancreatin in percentage maintains the same sequence in the samples under study as it was in the decrease in the efficacy of chymotrypsin and the total proteolytic efficacy, but it is smaller. The decrease in the efficacy of pure enzyme is, similarly to chymotrypsin, greater than the decrease in the efficacy of the enzyme in pancreatin. The present ballast substances thus significantly influence stability. (author)

  10. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  11. Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

    International Nuclear Information System (INIS)

    Dickinson, D.B.

    1975-01-01

    Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

  12. Multi-enzyme catalyzed processes: Next generation biocatalysis

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia; Sin, Gürkan; Gernaey, Krist

    2011-01-01

    Biocatalysis has been attracting increasing interest in recent years. Nevertheless, most studies concerning biocatalysis have been carried out using single enzymes (soluble or immobilized). Currently, multiple enzyme mixtures are attractive for the production of many compounds at an industrial...

  13. Catabolite repression of enzyme synthesis does not prevent sporulation.

    OpenAIRE

    Lopez, J M; Uratani-Wong, B; Freese, E

    1980-01-01

    In the presence of excess glucose, a decrease of guanine nucleotides in Bacillus subtilis initiated sporulation but did not prevent catabolite repression of three enzymes. Therefore, the ultimate mechanism(s) repressing enzyme synthesis differs from that suppressing sporulation.

  14. Pathogenicity and cell wall-degrading enzyme activities of some ...

    African Journals Online (AJOL)

    Dr. J. T. Ekanem

    2005-12-17

    Dec 17, 2005 ... be attributed to the activities of these cell wall degrading enzymes. Keywords: Cowpea ... bacteria have long been known to produce enzymes capable of ... Inoculated seeds were sown in small plastic pots filled with steam- ...

  15. Directing filtration to optimize enzyme immobilization in reactive membranes

    DEFF Research Database (Denmark)

    Luo, Jianquan; Marpani, Fauziah; Brites, Rita

    2014-01-01

    enzymatic reaction efficiency were evaluated in terms of enzyme loading, conversion rate and biocatalytic stability. Alcohol dehydrogenase (ADH) was selected as a model enzyme. Lower pressure, higher enzyme concentration and lower pH resulted in higher irreversible fouling resistance and lower permeate flux....... High pH during immobilization produced increased permeate flux but declines in conversion rates, likely because of the weak immobilization resulting from strong electrostatic repulsion between enzymes and membrane. The results showed that pore blocking as a fouling mechanism permitted a higher enzyme...... loading but generated more permeability loss, while cake layer formation increased enzyme stability but resulted in low loading rate. Low pH (near isoelectric point) favored hydrophobic and electrostatic adsorption of enzymes on the membrane, which reduced the enzyme stability. Neutral pH, however...

  16. Transcriptional regulation of the xylanolytic enzyme system of Aspergillus

    NARCIS (Netherlands)

    Peij, van N.N.M.E.

    1999-01-01

    Filamentous fungi, such as Aspergillus niger , produce high levels of polysaccharide degrading enzymes and are frequently used as production organisms for industrial enzyme preparations. The application of these polysaccharidases as xylanases and cellulases comprises

  17. Production and optimization of ligninolytic enzymes by white rot ...

    African Journals Online (AJOL)

    Production and optimization of ligninolytic enzymes by white rot fungus Schizophyllum ... size and nutritional factors (carbon and nitrogen ratio, mediators and metal ions). ... scale production of these enzymes for diverse industrial applications.

  18. Selected soil enzymes: Examples of their potential roles in the ...

    African Journals Online (AJOL)

    SERVER

    2008-02-05

    Feb 5, 2008 ... Soil enzymes regulate ecosystem functioning and in particular play a key role in nutrient cycling. In ... A better understanding of the role of these soil enzyme- es activity ..... measure of any disruption caused by pesticides, trace.

  19. ligninolytic enzymes of the fungus isolated from soil contaminated

    African Journals Online (AJOL)

    FUTE

    aimed at isolating lignin degrading fungi from soil contaminated with cow dung ... strain was screened for production of ligninolytic enzymes using Rhemazol Brilliant blue R ... put in airtight plastic bag and carried out to ..... Enzyme Microbial.

  20. Structure and function of α-glucan debranching enzymes

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Henriksen, Anette; Svensson, Birte

    2016-01-01

    α-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α......-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12–14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains......_39 enzymes could represent a “missing link” between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference....

  1. Discovery of enzymes for toluene synthesis from anoxic microbial communities

    DEFF Research Database (Denmark)

    Beller, Harry R.; Rodrigues, Andria V.; Zargar, Kamrun

    2018-01-01

    Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes...... phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from...... a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic...

  2. Preparation of immobilized enzyme membrane by radiation-cast-polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1989-01-01

    The preparation of immobilized enzyme membranes was studied by radiation cast-polymerization at low temperatures using cellulase enzyme, hydrophilic and hydrophobic monomers. The enzyme activity of the membranes was affected by monomer concentration, membrane thickness, and hydrophilicity of monomer, in which the membranes with 100 μm thickness from high monomer concentration (80%) had high enzyme activity, which was similar to that of the membranes with 1.0 mm thickness from low monomer concentration (20%). (author)

  3. Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

    Science.gov (United States)

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

    2011-02-17

    Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

  4. Studies on the enzymes produced by Basidiomycetes. Part 1. The production of crude enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Hong, J. S.; Kim, D.H.

    1981-01-01

    Cellulase, protease, and xylanase, formation by the basidiomycetes, Pleurotus ostreatus 301 and Lentinus edodes 3-1 in growth on rice straw medium were studied. Cultural conditions adequate for enzyme production and effects of various materials and inorganic salts added to the rice straw media were investigated. Lentinus edodes 3-1 was an excellent producer of cellulase and xylanase, and Pleurotus ostreatus 301 of protease. The optimum conditions for enzyme production were 30 degrees for cellulase production and at 25 degrees for xylanase and protease production, with 75% moisture content and initial pH of 5.0-6.0. The appropriate incubation times for enzyme production were 30 days and 35 days for Pleurotus ostreatus 301 and Lentinus edodes 3-1, respectively. Among the various materials added, defatted soybean, defatted rape seed, or defatted sesame were all effective in enzyme production but reduced mycelial growth. Rice bran was also effective, particularly at a 30% concentration. The addition of inorganic salts enhanced enzyme production. Among inorganic salts, the optimum concentration of CaCO3 was 5%, and that of CaSO4 was 2%.

  5. Mycelial growth interactions and mannan-degrading enzyme ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... enzymes (Frost and Moss, 1987). However, microbial enzymes are more in use due to cheaper substrates and ease of process modification. In microbial enzyme and biomass production, defined mixed culture method in which more than one organism grows simultaneously can result in increased biomass ...

  6. Microbial production of raw starch digesting enzymes | Sun | African ...

    African Journals Online (AJOL)

    Raw starch digesting enzymes refer to enzymes that can act directly on raw starch granules below the gelatinization temperature of starch. With the view of energy-saving, a worldwide interest has been focused on raw starch digesting enzymes in recent years, especially since the oil crisis of 1973. Raw starch digesting ...

  7. Development of the Enzyme-Substrate Interactions Concept Inventory

    Science.gov (United States)

    Bretz, Stacey Lowery; Linenberger, Kimberly J.

    2012-01-01

    Enzyme function is central to student understanding of multiple topics within the biochemistry curriculum. In particular, students must understand how enzymes and substrates interact with one another. This manuscript describes the development of a 15-item Enzyme-Substrate Interactions Concept Inventory (ESICI) that measures student understanding…

  8. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  9. Production of cell wall enzymes in pepper seedlings, inoculated with ...

    African Journals Online (AJOL)

    Pepper seedlings inoculated with arbuscular mycorrhizal AM fungus, Glomus etunicatum, produced cellulase, polygal-acturonase and pectin methylestrase enzymes. The activities of the enzymes increased as the pepper seedlings matured in age, showing that the activity of the enzymes in the seedlings was age mediated.

  10. Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes

    DEFF Research Database (Denmark)

    Krogh, Kristian Bertel Rømer; Mørkeberg, Astrid; Frisvad, Jens Christian

    2004-01-01

    For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commenly used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamentous fungi from ...

  11. A virus-based single-enzyme nanoreactor

    NARCIS (Netherlands)

    Comellas Aragones, M.; Engelkamp, H.; Claessen, V.I.; Sommerdijk, N.A.J.M.; Rowan, A.E.; Christianen, P.C.M.; Maan, J.C.; Verduin, B.J.M.; Cornelissen, J.J.L.M.; Nolte, R.J.M.

    2007-01-01

    Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or

  12. Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

    International Nuclear Information System (INIS)

    Korecka, Lucie; Jezova, Jana; Bilkova, Zuzana; Benes, Milan; Horak, Daniel; Hradcova, Olga; Slovakova, Marcela; Viovy, Jean-Louis

    2005-01-01

    The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized

  13. Kininase enzymes of cat eye tissues

    International Nuclear Information System (INIS)

    Ryan, J.W.; Anderson, D.R.

    1986-01-01

    Eye tissues contain kininase activities, including an angiotensin converting enzyme (ACE)-like activity. The authors have begun further to characterize the ACE-like activity and to examine for another reputed kininase, carboxypeptidase N (CPN). Homogenates of tissues of 6 cat eyes and paired plasmas were assayed for ACE using 3 acyl-tripeptide substrates, 3 H-benzoylated F-A-P, F-G-P and A-G-P (respectively, BFAP, BFGP and BAGP). CPN was assayed using 3 H-benzoyl-A-R. All eye tissues and fluids contained ACE- and CPN-like activities. The ACE activity was clearly owing to ACE: relative values of Kc/Km for BFAP, BFGP and BAGP were those for pure ACE (2.213, 1.751 and 1.0); reactivities with inhibitors were as expected (Ki for captopril, MK 422 and RAC-X-65: 2.7, 0.62 and 0.31 nM). EDTA inhibited both ACE and CPN (I 50 's: 43 and 47 μM). CPN activity was inhibited by 2-mercaptomethyl-3-guanidinoethylthiopropionate (Ki 2.4 nM). However, distributions of the two enzymes differed markedly. Virtually all tissues contained ACE at specific activities higher than that of plasma. Specific activities appeared to be a function of tissue vascularity (for choroid, ciliary body, iris, retina and plasma: 7.31, 2.57, 1.98, 1.53 and 0.21 pmol/mg protein). Only iris contained more CPN that did plasma (23.0 v. 7.21 pmol/mg protein). The tissue distribution of ACE is that expected for an endothelial-associated enzyme. Plasma may be the major source of CPN in eye tissues other than iris

  14. Enzyme-potentiated desensitization in otolaryngic allergy.

    Science.gov (United States)

    Pulec, Jack L

    2002-03-01

    This is a preliminary report of a new method of treating otolaryngic allergy with enzyme-potentiated desensitization (EPD). The nature of EPD and its use in otolaryngology are described. Thirty-six patients have been treated and followed in a private medical practice since February 1997. This article reviews the clinical features of EPD and provides six cases as examples; the clinical features described include allergic rhinitis, serous otitis media, asthma, dermatitis, fixed food allergy, and Ménière's disease. EPD is an effective technique for the treatment of otolaryngic allergy and offers advantages over conventional techniques.

  15. NADPH oxidase: an enzyme for multicellularity?

    Science.gov (United States)

    Lalucque, Hervé; Silar, Philippe

    2003-01-01

    Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

  16. Expression of Enzymes that Metabolize Medications

    Science.gov (United States)

    Wotring, Virginia E.; Peters, C. P.

    2012-01-01

    Most pharmaceuticals are metabolized by the liver. Clinically-used medication doses are given with normal liver function in mind. A drug overdose can result if the liver is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism we want to understand the effects of spaceflight on the enzymes of the liver.

  17. The enzymes of bacterial census and censorship.

    Science.gov (United States)

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Analysis of serum angiotensin-converting enzyme.

    Science.gov (United States)

    Muller, B R

    2002-09-01

    Serum angiotensin-converting enzyme (SACE) levels are influenced by genetic polymorphism. Interpretation of serum levels with the appropriate genotypic reference range improves the diagnostic sensitivity of the assay for sarcoidosis. SACE assays are performed by a large number of routine clinical laboratories. However, there is no external quality assessment (EQA) for SACE other than an informal regional scheme. This showed analytical performance of SACE assays to be poor, with a diversity of reference ranges, leading to widely disparate clinical classification of EQA samples. Genetic polymorphism combined with poor analytical performance suggest that perhaps SACE assays should revert to being the province of specialized laboratories.

  19. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

    Directory of Open Access Journals (Sweden)

    Daryl L Moorhead

    2013-08-01

    Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

  20. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    Science.gov (United States)

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.