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Sample records for enzymatic activities consistent

  1. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes.

  2. Extracellular enzymatic activity of Microsporum canis isolates.

    PubMed

    Papini, R; Mancianti, F

    The enzymatic activity of 70 feline and canine Microsporum canis isolates was determined by the Api-Zym test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity of M. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted by M. canis.

  3. Non-ionic surfactants do not consistently improve the enzymatic hydrolysis of pure cellulose.

    PubMed

    Zhou, Yan; Chen, Hongmei; Qi, Feng; Zhao, Xuebing; Liu, Dehua

    2015-04-01

    Non-ionic surfactants have been frequently reported to improve the enzymatic hydrolysis of pretreated lignocellulosic biomass and pure cellulose. However, how the hydrolysis condition, substrate structure and cellulase formulation affect the beneficial action of surfactants has not been well elucidated. In this work, it was found that the enzymatic hydrolysis of pure cellulose was not consistently improved by surfactants. Contrarily, high surfactant concentration, e.g. 5 g/L, which greatly improved the hydrolysis of dilute acid pretreated substrates, actually showed notable inhibition to pure cellulose conversion in the late phase of hydrolysis. Under an optimal hydrolysis condition, the improvement by surfactant was limited, but under harsh conditions surfactant indeed could enhance cellulose conversion. It was proposed that non-ionic surfactants could interact with substrates and cellulases to impact the adsorption behaviors of cellulases. Therefore, the beneficial action of surfactants on pure cellulose hydrolysis is influenced by hydrolysis condition, cellulose structural features and cellulase formulation.

  4. Enzymatic activities in coniferous leaf litter

    SciTech Connect

    Spalding, B.P.

    1980-07-01

    Assays for measuring the activities of cellulase, xylanase, mannase, amylase, ..beta..-glucosidase, invertase, and protease employing buffered suspensions of ground coniferous and deciduous leaf litter exhibited zero-order kinetics. Only a small percentage of the whole-litter activities of invertase, ..beta..-glucosidase, and protease were extractable into 0.05M potassium acetate, pH 5.0; however, extractable activities of cellulase and xylanase represented from 39 to 174% of the whole-litter activities indicating their soluble exocellar nature. Extractable protease and amylase activities were best correlated with the average daily rates of CO/sub 2/ evolution in a group of 90 leaf litter samples equally representing 18 coniferous species. Enzymatic activities were readily detectable in extracts of all samples but classification of the samples by species provided little differentiation in the distribution of either enzymatic activities or rates of CO/sub 2/ evolution. Mannase, cellulase, and xylanase activities were well-correlated with each other in all samples. Assays attempting to measure a pool of readily-metabolizable substances in litter by extractable reducing substances, ninhydrin-positive substances, glucose, and phenolics failed to show correlation coefficients >0.41 with rates of CO/sub 2/ evolution. Addition of D-(+)-catechin to litter extracts, up to levels equivalent to those observed in the group of samples, did not inhibit any carbohydrase thus suggesting the lack of inhibition of litter-decomposing enzymes by the concentrations of phenolics present in these coniferous leaf litters.

  5. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  6. [Histidine triad protein superfamily--biological function and enzymatic activity].

    PubMed

    Krakowiak, Agnieszka; Fryc, Izabela

    2012-01-01

    The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

  7. Enzymatic activity of fungi isolated from crops

    PubMed Central

    Cholewa, Grażyna; Sobczak, Paweł; Silny, Wojciech; Nadulski, Rafał; Wojtyła-Buciora, Paulina; Zagórski, Jerzy

    2016-01-01

    Aim To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye). Isolated strains were also classified in the scale of biosafety levels (BSL). Material and methods The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil) collected in the Lublin region (eastern Poland). API ZYM test (bioMérieux) was carried out according to the manufacturer’s instructions. Classification of BSL (Biosafety levels) was based on the current literature. Results High enzymatic activity was found in strains cultured in media containing 1% of wheat grain (Bipolaris holmi, Penicillium decumbens) and with an addition of 1% of rye grain (Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata). The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used. Conclusions Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes. PMID:28035224

  8. Changes in enzymatic activity in composts containing chicken feathers.

    PubMed

    Bohacz, Justyna; Korniłłowicz-Kowalska, Teresa

    2009-07-01

    Enzymatic activity, i.e. respiratory activity, dehydrogenase activity, phosphatase activity, caseinian protease activity, BAA protease activity and urease activity, was determined to investigate the process of biochemical transformations and to select enzymatic indices of maturity of composts prepared from feathers and lignocellulose wastes (bark, straw). Composting was conducted for 7 months, with periodic determinations of activity of the enzymes. The study revealed significant differences in the enzymatic activity, related with the duration of composting and with the substrate composition of the composts. Generally, composts enriched with straw were characterised by higher enzymatic activity than composts without any addition of straw. It was found that the activity of such enzymes as cellulase and protease, towards the end of the period of composting decreased and stabilised. The enzymes enumerated can be taken into consideration in estimation of the maturity of composts prepared from feathers and lignocellulose wastes.

  9. Enzymatic activity preservation and protection through entrapment within degradable hydrogels.

    PubMed

    Mariani, Angela M; Natoli, Mary E; Kofinas, Peter

    2013-11-01

    This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work β-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N'-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create "biogels." Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks , decreased when increasing the crosslink density from 78.6 to 14.7 min⁻¹ over a range of 1-20 mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10 mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40 mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their

  10. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  11. A general framework for thermodynamically consistent parameterization and efficient sampling of enzymatic reactions.

    PubMed

    Saa, Pedro; Nielsen, Lars K

    2015-04-01

    Kinetic models provide the means to understand and predict the dynamic behaviour of enzymes upon different perturbations. Despite their obvious advantages, classical parameterizations require large amounts of data to fit their parameters. Particularly, enzymes displaying complex reaction and regulatory (allosteric) mechanisms require a great number of parameters and are therefore often represented by approximate formulae, thereby facilitating the fitting but ignoring many real kinetic behaviours. Here, we show that full exploration of the plausible kinetic space for any enzyme can be achieved using sampling strategies provided a thermodynamically feasible parameterization is used. To this end, we developed a General Reaction Assembly and Sampling Platform (GRASP) capable of consistently parameterizing and sampling accurate kinetic models using minimal reference data. The former integrates the generalized MWC model and the elementary reaction formalism. By formulating the appropriate thermodynamic constraints, our framework enables parameterization of any oligomeric enzyme kinetics without sacrificing complexity or using simplifying assumptions. This thermodynamically safe parameterization relies on the definition of a reference state upon which feasible parameter sets can be efficiently sampled. Uniform sampling of the kinetics space enabled dissecting enzyme catalysis and revealing the impact of thermodynamics on reaction kinetics. Our analysis distinguished three reaction elasticity regions for common biochemical reactions: a steep linear region (0> ΔGr >-2 kJ/mol), a transition region (-2> ΔGr >-20 kJ/mol) and a constant elasticity region (ΔGr <-20 kJ/mol). We also applied this framework to model more complex kinetic behaviours such as the monomeric cooperativity of the mammalian glucokinase and the ultrasensitive response of the phosphoenolpyruvate carboxylase of Escherichia coli. In both cases, our approach described appropriately not only the kinetic

  12. Trisomy 21 consistently activates the interferon response.

    PubMed

    Sullivan, Kelly D; Lewis, Hannah C; Hill, Amanda A; Pandey, Ahwan; Jackson, Leisa P; Cabral, Joseph M; Smith, Keith P; Liggett, L Alexander; Gomez, Eliana B; Galbraith, Matthew D; DeGregori, James; Espinosa, Joaquín M

    2016-07-29

    Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.

  13. Enzymatic activity of allergenic house dust and storage mite extracts.

    PubMed

    Morales, Maria; Iraola, Víctor; Leonor, Jose R; Carnés, Jerónimo

    2013-01-01

    Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy.

  14. Effects of organic carbon sequestration strategies on soil enzymatic activities

    NASA Astrophysics Data System (ADS)

    Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.

    2009-04-01

    Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

  15. Trisomy 21 consistently activates the interferon response

    PubMed Central

    Sullivan, Kelly D; Lewis, Hannah C; Hill, Amanda A; Pandey, Ahwan; Jackson, Leisa P; Cabral, Joseph M; Smith, Keith P; Liggett, L Alexander; Gomez, Eliana B; Galbraith, Matthew D; DeGregori, James; Espinosa, Joaquín M

    2016-01-01

    Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.001 PMID:27472900

  16. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

  17. Enzymatic and Non-Enzymatic Virulence Activities of Dermatophytes on Solid Media

    PubMed Central

    Elavarashi, Elangovan; Rangarajan, Sudha

    2017-01-01

    Introduction Dermatophytes are keratinophilic fungi causing superficial cutaneous infections that account 20-25% of the global population. As per literature search, there is a dearth in the study on virulence factors of dermatophytes from the Indian sub-continent and moreover the association of the virulence factors and the host tissue in vitro helps in understanding the host-pathogen interaction. Aim To analyse the enzymatic and non-enzymatic virulence activities of dermatophytes on solid media. Materials and Methods A total of 11 isolates, three standard American Type Culture Collection (ATCC) strains- Trichophyton rubrum- 28188, Trichophyton mentagrophytes- 9533, Trichophyton tonsurans- 28942, one CBS KNAW Fungal Biodiversity Centre strain- Arthroderma grubyi- 243.66, five clinical isolates- T. rubrum, T. mentagrophytes, Trichophyton rubrum var. raubitschekii, Trichophyton interdigitale, Epidermophyton floccosum, and two laboratory isolates - Microsporum gypseum and Microsporum canis were screened for the production of virulence enzymes such as phospholipase, lipase, protease, gelatinase and non-enzyme virulence factors (haemolytic activity) of dermatophytes. The clinical isolates were identified from a tertiary care hospital, Chennai. These dermatophytes were tested upon specific substrates on solid media such as egg yolk, tween 80, bovine serum albumin, gelatin powder and sheep blood respectively. Results The virulence activity of phospholipase, lipase, protease and gelatinase was observed from all the dermatophyte species. T. rubrum, T. rubrum ATCC strain, T. rubrum var. raubitschekii, T. mentagrophytes, T. mentagrophytes ATCC strain, T. interdigitale and A. grubyi CBS strain produced complete haemolysis, whereas other dermatophytes showed no haemolytic activity. Conclusion Phospholipase, lipase, protease and gelatinase act as enzymatic virulence marker and the T. rubrum complex, T. mentagrophytes complex and A. grubyi showed complete haemolysis and hence

  18. Production of Enzymatically Active Human Acetylcholinesterase in E. Coli

    DTIC Science & Technology

    1993-10-01

    AD-A282 703 lE1l1lm11I AD( CONTRACT NO: DAMD17-90-C-0107 TITLE: PRODUCTION OF ENZYMATICALLY ACTIVE HUMAN ACETYLCHOLINESTERASE IN E . COLI PRINCIPAL...FUNDING NUMBERS Production of Enzymatically Active Human Contract No. Acetylcholinesterase in E . coli DAMD17-90-C-0107 6. AUTHOR(S) M. Gorecki, Ph.D. and M...S493pMFL-52Ser - Run #1 37 Table 8: Summary of reconstitution and purification of rhAChE derived from E . coli S493pMFL-52Ser - Run #2 38 Table 9

  19. Enzymatic activity of rodents acclimated to cold and long scotophase

    NASA Astrophysics Data System (ADS)

    Fourie, F. Le R.; Haim, A.

    1980-09-01

    Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

  20. Macrophage migration inhibitory factor (MIF) enzymatic activity and lung cancer.

    PubMed

    Mawhinney, Leona; Armstrong, Michelle E; O' Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2015-04-16

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif (P1G)). Primary tumor growth was significantly attenuated in both Mif-KO and Mif (P1G) mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.

  1. Biological activity of camel milk casein following enzymatic digestion.

    PubMed

    Salami, Maryam; Moosavi-Movahedi, Ali Akbar; Moosavi-Movahedi, Faezeh; Ehsani, Mohammad Reza; Yousefi, Reza; Farhadi, Mohammad; Niasari-Naslaji, Amir; Saboury, Ali Akbar; Chobert, Jean-Marc; Haertlé, Thomas

    2011-11-01

    The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.

  2. Some enzymatic activities associated with purified parapoxvirions.

    PubMed Central

    Caplen, H S; Holowczak, J A

    1983-01-01

    Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons. Images PMID:6188861

  3. Mechanobiocatalysis: Modulating Enzymatic Activity with Mechanical Force

    DTIC Science & Technology

    2015-09-28

    displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers resulted in...intrinsic activities displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers ...of eYFP-containing polymer composites via the application of mechanical force, as well as showing that the photophysical properties displayed by

  4. Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

    PubMed

    Vassel, N; Cox, C D; Naseem, R; Morse, V; Evans, R T; Power, R L; Brancale, A; Wann, K T; Campbell, A K

    2012-01-01

    Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

  5. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.

    PubMed

    Ahmed, Rachel R; Holler, Christopher J; Webb, Robin L; Li, Feng; Beckett, Tina L; Murphy, M Paul

    2010-02-01

    beta-Secretase is the rate limiting enzymatic activity in the production of the amyloid-beta peptide (Abeta) and is thought to be involved in Alzheimer's disease (AD) pathogenesis. Although BACE1 (beta-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Abeta, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Abeta concentration. These data indicate that BACE1 likely accounts for most of the Abeta produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease.

  6. First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT.

    PubMed

    Bouvier, Emmanuel; Thirot, Sylvie; Schmidt, Frédéric; Monneret, Claude

    2004-03-01

    Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT. They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the spacer substitution. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics. As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological evaluations.

  7. Efficient enzymatic acrylation through transesterification at controlled water activity.

    PubMed

    Nordblad, Mathias; Adlercreutz, Patrick

    2008-04-15

    Enzymatic acrylation is a process of potentially strong interest to the chemical industry. Direct esterification involving acrylic acid is unfortunately rather slow, with inhibition phenomena appearing at high acid concentrations. In the present study the acrylation of 1-octanol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was shown to be as much as an order of magnitude faster when ethyl acrylate served as the donor of the acrylic group. Water activity is a key parameter for optimizing the rate of ester synthesis. The optimum water activity for the esterification of octanol by acrylic acid was found to be 0.75, that for its esterification by propionic acid to be 0.45 and the transesterification involving ethyl acrylate to be fastest at a water activity of 0.3. The reasons for these differences in optimum water activity are discussed in terms of enzyme specificity, substrate solvation, and mass transfer effects.

  8. Enzymatic Activity of the Scaffold Protein Rapsyn for Synapse Formation.

    PubMed

    Li, Lei; Cao, Yu; Wu, Haitao; Ye, Xinchun; Zhu, Zhihui; Xing, Guanglin; Shen, Chengyong; Barik, Arnab; Zhang, Bin; Xie, Xiaoling; Zhi, Wenbo; Gan, Lin; Su, Huabo; Xiong, Wen-Cheng; Mei, Lin

    2016-12-07

    Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.

  9. A designed supramolecular protein assembly with in vivo enzymatic activity.

    PubMed

    Song, Woon Ju; Tezcan, F Akif

    2014-12-19

    The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

  10. Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

    SciTech Connect

    Dabulis, K.; Klibanov, A.M. )

    1993-03-05

    When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH[sub 2], their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient-activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its regular' counterpart.

  11. Enzymatic activity preservation through entrapment within degradable hydrogel networks

    NASA Astrophysics Data System (ADS)

    Mariani, Angela Marie

    This dissertation aimed to design and develop a "biogel;" a reproducible, abiotic, and biocompatible polymer hydrogel matrix, that prolongs enzymatic stability allowing for rapid production of biomolecules. The researched entrapment method preserves enzyme activity within an amicable environment while resisting activity reduction in the presence of increased pH environmental challenges. These biogels can be used in a number of applications including repeated production of small molecules and in biosensors. Five main objectives were accomplished: 1) Biogels capable of maintaining enzymatic functionality post-entrapment procedures were fabricated; 2) Biogel activity dependence on crosslinker type and crosslink density was determined; 3) Biogel composition effects on sustained activity after storage were compared; 4) Biogel activity dependence on charged monomer moieties was evaluated, and 5) Combined optimization knowledge gained from the first four objectives was utilized to determine the protection of enzymes within hydrogels when challenged with an increased pH above 8. Biogels were fabricated by entrapping β-galactosidase (lactase) enzyme within acrylamide (ACR) gels crosslinked with poly(ethylene glycol) diacrylate (PEGDA, degradable through hydrolysis) or N,N'-methylenebisacrylamide (BIS, non-degradable). Initial hydrogel entrapment reduced activity to 40% in ACR/PEGDA gels, compared to a 75% reduction in initial activity of ACR/BIS biogels. Once entrapped, these enzymes resist activity reduction in the presence of environmental challenges, such as altering the pH from 7 to above 8. When biogels were challenged at a pH of 8, activity retention positively correlated to PEGDA crosslinker density; increasing from 48% to 91% retention in 30 to 40 mole % PEGDA biogels as compared to solution based control which retained only 23%. Retention of activity when perturbed from pH 7 is advantageous for biogel applications including the repeated production of desired small

  12. The enzymatic activity from the sediment of the Gilau dam reservoir - Cluj county.

    PubMed

    Curticapean, Manuela-Claudia; Dragan-Bularda, Mihail

    2007-01-10

    The enzymological studies on the sediment of the accumulation lake that has the main purpose of supplying drinking water to the city of Cluj-Napoca and the nearby villages, were aimed at the comprehensive understanding of the complex processes that happen in these habitats of special significance. In the sediment samples the following enzymatic activities have been quantitatively determined: phosphatase, actual and potential dehydrogenase, catalase, urease and protease. Non-enzymatic catalytic activity was also measured. Based on the relative values for the enzymatic activities, the enzymatic indicator of the sediment quality (EISQ) was calculated (ranging from 0.1 to 0.7). The enzymatic activities have been qualitatively determined for maltase, saccharase, lactase, cellobiase, amylase, dextranase, levanase, cellulase and inulinase. The correlation between the enzymatic and bacteriologic potential was statistically calculated.

  13. Nanoparticle-mediated remote control of enzymatic activity.

    PubMed

    Knecht, Leslie D; Ali, Nur; Wei, Yinan; Hilt, J Zach; Daunert, Sylvia

    2012-10-23

    Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide cross-linked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HAD(ST). This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe(3)O(4) nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion-limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields.

  14. Size-consistent self-consistent configuration interaction from a complete active space

    NASA Astrophysics Data System (ADS)

    Ben Amor, Nadia; Maynau, Daniel

    1998-04-01

    The size-consistent self-consistent (SC) 2 method is based on intermediate Hamiltonians and ensures size-extensivity of any configuration interaction (CI) by correcting its diagonal elements. In this work, an (SC) 2 dressing is proposed on a complete active space SDCI. This approach yields a more efficient code which can treat larger multireference problems. Tests are proposed on the potential energy curve of F 2, the bond stretching of water and the inclusion of an Be atom in the H 2 molecule. Comparisons with approximate methods such as average quadratic coupled cluster (AQCC) are presented. AQCC appears as a good approximation to (SC) 2.

  15. Comparison of lab, pilot, and industrial scale low consistency mechanical refining for improvements in enzymatic digestibility of pretreated hardwood.

    PubMed

    Jones, Brandon W; Venditti, Richard; Park, Sunkyu; Jameel, Hasan

    2014-09-01

    Mechanical refining has been shown to improve biomass enzymatic digestibility. In this study industrial high-yield sodium carbonate hardwood pulp was subjected to lab, pilot and industrial refining to determine if the mechanical refining improves the enzymatic hydrolysis sugar conversion efficiency differently at different refining scales. Lab, pilot and industrial refining increased the biomass digestibility for lignocellulosic biomass relative to the unrefined material. The sugar conversion was increased from 36% to 65% at 5 FPU/g of biomass with industrial refining at 67.0 kWh/t, which was more energy efficient than lab and pilot scale refining. There is a maximum in the sugar conversion with respect to the amount of refining energy. Water retention value is a good predictor of improvements in sugar conversion for a given fiber source and composition. Improvements in biomass digestibility with refining due to lab, pilot plant and industrial refining were similar with respect to water retention value.

  16. Controlling enzymatic activity by immobilization on graphene oxide.

    PubMed

    Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P

    2017-04-01

    In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

  17. Enzymatic activities in limb muscles subjected to external fixation with ring-hybrid frames.

    PubMed

    Reznick, Abraham Z; Coleman, Raymond; Stein, Haim

    2007-04-01

    Enzymatic activities, which originate in the muscle envelope of tibiae with an experimental segmental bone loss, provide additional evidence for the intimate bone-muscle interrelationships in new bone formation.

  18. Enzymatic activity inside and outside of water-stable aggregates in soils under different land use

    NASA Astrophysics Data System (ADS)

    Garbuz, S. A.; Yaroslavtseva, N. V.; Kholodov, V. A.

    2016-03-01

    A method is presented for assessing the distribution of enzymatic activity inside and outside of water-stable aggregates. Two samples of water-stable aggregates >1 mm have been isolated from dry aggregates of 1-2 mm. To determine the enzymatic activity, a substrate has been added to one of the samples without disaggregation; the other sample has been preliminarily disaggregated. Enzymatic activity within waterstable aggregates has been assessed from the difference between the obtained results under the supposition that the penetration of substrate within the water-saturated aggregates is hampered, and enzymatic reactions occur only at the periphery. The levels and distributions of enzymatic (peroxidase, polyphenol oxidase, and catalase) activities in water-stable aggregates of soddy-podzolic soils under forest and plowland and typical chernozems of long-term field experiments have been studied. The peroxidase, polyphenol oxidase, and catalase activities of water-stable aggregates vary from 6 to 23, from 7 to 30, and from 5 to 7 mmol/(g h), respectively. The ratio between the enzymatic activities inside and outside of soil aggregates showed a higher dependence on soil type and land use, as well as on the input of organic matter and the structural state, than the general activity level in water-stable aggregates.

  19. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement.

    PubMed

    Sun, Wenjie; Vallooran, Jijo J; Zabara, Alexandru; Mezzenga, Raffaele

    2014-06-21

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.

  20. Adsorption-induced changes in ribonuclease A structure and enzymatic activity on solid surfaces.

    PubMed

    Wei, Yang; Thyparambil, Aby A; Wu, Yonnie; Latour, Robert A

    2014-12-16

    Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy.

  1. A water activity control system for enzymatic reactions in organic media.

    PubMed

    Petersson, Anna E V; Adlercreutz, Patrick; Mattiasson, Bo

    2007-06-01

    A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.

  2. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  3. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    PubMed Central

    Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    simulations were consistent with these observations. Conclusions: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies. PMID:23668778

  4. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement

    NASA Astrophysics Data System (ADS)

    Sun, Wenjie; Vallooran, Jijo J.; Zabara, Alexandru; Mezzenga, Raffaele

    2014-05-01

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them

  5. Differential enzymatic activity of common haplotypic versions of the human acidic Mammalian chitinase protein.

    PubMed

    Seibold, Max A; Reese, Tiffany A; Choudhry, Shweta; Salam, Muhammad T; Beckman, Kenny; Eng, Celeste; Atakilit, Amha; Meade, Kelley; Lenoir, Michael; Watson, H Geoffrey; Thyne, Shannon; Kumar, Rajesh; Weiss, Kevin B; Grammer, Leslie C; Avila, Pedro; Schleimer, Robert P; Fahy, John V; Rodriguez-Santana, Jose; Rodriguez-Cintron, William; Boot, Rolf G; Sheppard, Dean; Gilliland, Frank D; Locksley, Richard M; Burchard, Esteban G

    2009-07-17

    Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.

  6. Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation

    PubMed Central

    Wang, Yuanyuan; Sung, Chun Chau; Chung, Kenny K. K.

    2017-01-01

    Tyrosine hydroxylase (TH) is a rate-limiting step enzyme in the synthesis of catecholamines. Catecholamines function both as hormone and neurotransmitters in the peripheral and central nervous systems, therefore TH’s expression and enzymatic activity is tightly regulated by various mechanisms. Several post-translational modifications have been shown to regulate TH’s enzymatic activity such as phosphorylation, nitration and S-glutathionylation. While phosphorylation at N-terminal of TH can activate its enzymatic activity, nitration and S-glutathionylation can inactivate TH. In this study, we found that TH can also be S-nitrosylated by nitric oxide (NO). S-nitrosylation is a reversible modification of cysteine (cys) residue in protein and is known to be an emerging signaling mechanism mediated by NO. We found that TH can be S-nitrosylated at cys 279 and TH S-nitrosylation enhances its enzymatic activity both in vitro and in vivo. These results provide a novel mechanism of how NO can modulate TH’s enzymatic activity through S-nitrosylation. PMID:28287127

  7. Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction.

    PubMed

    Karnaouri, Anthi C; Topakas, Evangelos; Christakopoulos, Paul

    2014-01-01

    An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme.

  8. Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus.

    PubMed

    Hu, Feng; Zhao, Yan; Qi, Xiaole; Cui, Hongyu; Gao, Yulong; Gao, Honglei; Liu, Changjun; Wang, Yongqiang; Zhang, Yanping; Li, Kai; Wang, Xiaomei; Wang, Yunfeng

    2015-10-01

    The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses.

  9. Enzymatic activity regulated by a surfactant and hydroxypropyl β-cyclodextrin.

    PubMed

    Li, Qingzhong; Zhai, Tao; Du, Kun; Li, Yanxin; Feng, Wei

    2013-12-01

    Circular dichroism spectra reveal that sodium dodecyl benzene sulfonate (SDBS) at low concentrations can effectively prevent the aggregation of lysozyme molecules, while SDBS at high concentrations can lead to conformational and structural change of the protein. SDBS is able to inhibit the enzymatic activity of lysozyme in a highly efficient dose-dependent manner. The interaction mechanism of SDBS with lysozyme has been investigated by measuring optical spectra. Based on fluorescence and UV-vis spectra, microenvironmental change in and around the active site region induced by SDBS has been revealed and explained. Two-dimensional FTIR spectra have been analyzed to identify the secondary structures and residues of lysozyme, which have a preferential interaction with SDBS. Hydroxypropyl β-cyclodextrin (HP-β-CD) was used to detach SDBS from the inactivated enzyme, and complete recovery of enzymatic activity was achieved. Thus, the enzymatic activity of lysozyme can be regulated by SDBS and HP-β-CD.

  10. Biologically Active Oxylipins from Enzymatic and Nonenzymatic Routes in Macroalgae

    PubMed Central

    Barbosa, Mariana; Valentão, Patrícia; Andrade, Paula B.

    2016-01-01

    Marine algae are rich and heterogeneous sources of great chemical diversity, among which oxylipins are a well-recognized class of natural products. Algal oxylipins comprise an assortment of oxygenated, halogenated, and unsaturated functional groups and also several carbocycles, varying in ring size and position in lipid chain. Besides the discovery of structurally diverse oxylipins in macroalgae, research has recently deciphered the role of some of these metabolites in the defense and innate immunity of photosynthetic marine organisms. This review is an attempt to comprehensively cover the available literature on the chemistry, biosynthesis, ecology, and potential bioactivity of oxylipins from marine macroalgae. For a better understanding, enzymatic and nonenzymatic routes were separated; however, both processes often occur concomitantly and may influence each other, even producing structurally related molecules. PMID:26805855

  11. Effect of length of molecular recognition moiety on enzymatic activity switching.

    PubMed

    Oshiba, Yuhei; Tamaki, Takanori; Ohashi, Hidenori; Hirakawa, Hidehiko; Yamaguchi, Satoshi; Nagamune, Teruyuki; Yamaguchi, Takeo

    2013-10-01

    We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG₂-3mD conjugate, but was dramatically reduced in the biotin-PEG₁₁-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG₁₁, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG₂-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.

  12. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  13. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these enz...

  14. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these en...

  15. Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes.

    PubMed

    Bader, Kenneth B; Bouchoux, Guillaume; Peng, Tao; Klegerman, Melvin E; McPherson, David D; Holland, Christy K

    2015-08-01

    Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.

  16. A new biomimetic route to engineer enzymatically active mechano-responsive materials.

    PubMed

    Rios, César; Longo, Johan; Zahouani, Sarah; Garnier, Tony; Vogt, Cédric; Reisch, Andreas; Senger, Bernard; Boulmedais, Fouzia; Hemmerlé, Joseph; Benmlih, Karim; Frisch, Benoît; Schaaf, Pierre; Jierry, Loïc; Lavalle, Philippe

    2015-04-04

    Using modified β-galactosidase covalently linked to cross-linked polyelectrolyte multilayers (PEM), catalytically active materials have been designed. Their enzymatic activity can be modulated, partially in a reversible way, simply by stretching. This strategy, based on enzyme conformational changes, constitutes a new tool for the development of biocatalytic mechano-responsive materials.

  17. Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit.

    PubMed

    Fujii, Takuto; Watanabe, Midori; Shimizu, Takahiro; Takeshima, Hiroshi; Kushiro, Keiichiro; Takai, Madoka; Sakai, Hideki

    2016-06-01

    The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK.

  18. An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

    PubMed

    Zanin, Mark; Duan, Susu; Wong, Sook-San; Kumar, Gyanendra; Baviskar, Pradyumna; Collin, Emily; Russell, Charles; Barman, Subrata; Hause, Benjamin; Webby, Richard

    2017-01-15

    Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

  19. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    SciTech Connect

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-29

    An enzyme that converts (/sup 3/H, /sup 32/P)ATP, with a /sup 3/H:/sup 32/P ratio of 1:1, to oligoadenylates with the same /sup 3/H:/sup 32/P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of /sup 3/H:/sup 32/P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.

  20. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities.

    PubMed

    Devash, Y; Reichman, M; Sela, I; Reichenbach, N L; Suhadolnik, R J

    1985-01-29

    An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Inhibitory effect of nicotinamide on enzymatic activity of selected fungal strains causing skin infection.

    PubMed

    Ciebiada-Adamiec, Anna; Małafiej, Eugeniusz; Ciebiada, Ireneusz

    2010-05-01

    Pathogenicity of fungi is connected with their ability to easily penetrate the host tissues, survive in the infected host organism and use the elements of the host tissues as nutrients. Hence, the co-occurrence of pathogenic properties with the high enzymatic activity, which is manifested through the production of various enzymes including extracellular enzymes, was observed. It can be expected that it is possible to decrease fungal pathogenicity by lowering their enzymatic activity. The aim of the study was to determine the effect of nicotinamide on enzymatic activity of the fungi, which are most frequently isolated in cases of skin infection. Enzymatic activity was analysed using 15 Candida albicans, 15 Trichophyton rubrum and 15 Trichophyton mentagrophytes strains. The strains used for the study were collected from the current diagnostic material. API ZYM tests were used in diagnostic analysis. MICs of nicotinamide were determined by the macrodilution method in liquid medium. In the case of Candida strains, the presence of nicotinamide in the broth had a significant effect on the decrease of enzymatic activity (P < 0.05) of esterase (C4), esterase lipase (C-8), valin-arylamidase, acid phosphatase and alpha-glycosydase. A considerably stronger effect of nicotinamide was observed in the case of dermatophytes (P < 0.005). Its action led to a decrease in the activity of all the enzymes under study except alpha-glucosidase produced by T. rubrum strains. Thus, nicotinamide exhibited biological activity towards C. albicans, T. rubrum and Trichophyton mentagrophytes, which resulted in a decrease in the activity of enzymes produced by the fungi.

  2. ALDH enzymatic activity and CD133 positivity and response to chemotherapy in ovarian cancer patients.

    PubMed

    Ricci, Francesca; Bernasconi, Sergio; Porcu, Luca; Erba, Eugenio; Panini, Nicolò; Fruscio, Robert; Sina, Federica; Torri, Valter; Broggini, Massimo; Damia, Giovanna

    2013-01-01

    The prognostic/predictive role of both CD133 and Aldehyde dehydrogenase (ALDH) expression in human ovarian cancer remains elusive. This is an observational study that investigated the expression of CD133 and of ALDH enzymatic activity in fresh ovarian cancer samples and their association with different clinic-pathological patient' characteristics and explored their possible predictive/prognostic role. We analyzed the expression of CD133 and ALDH enzymatic activity in 108 human ovarian cancer samples. We found that among the total patients analyzed, 13% of them was completely negative for ALDH activity and 26% was negative for CD133 staining. Both markers were variably expressed within the samples and when both studied in the same tumor sample, no statistically significant correlation between ALDH enzymatic activity and CD133 expression was found. No statistical significant correlation was found also between the percentage values of positive ALDH and CD133 cells and the number of serial passages patient's cultures underwent, suggesting that these markers do not confer by themselves a self-renewal growth advantage to the cultures. Lower levels of CD133 were associated with higher tumor grade. No correlation with response to therapy, progression free survival and overall survival was found. Our data suggest that neither ALDH enzymatic activity nor CD133 expression provide additional predictive/prognostic information in ovarian cancer patients.

  3. The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

    NASA Astrophysics Data System (ADS)

    Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

    2017-03-01

    We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

  4. Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

    PubMed

    Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

    2015-02-11

    Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

  5. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance

    PubMed Central

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  6. Effect of restricted motion in high temperature on enzymatic activity of the pancreas

    NASA Technical Reports Server (NTRS)

    Abdusattarov, A.; Smirnova, G. I.

    1980-01-01

    Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

  7. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    NASA Technical Reports Server (NTRS)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  8. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity

    PubMed Central

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4–8 nm), strategy 2: self assembled monolayers (SAMs) (1–5 nm), strategy 3: polymer layer (1–5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions. PMID:21949594

  9. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity.

    PubMed

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4-8 nm), strategy 2: self assembled monolayers (SAMs) (1-5 nm), strategy 3: polymer layer (1-5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions.

  10. [Pharmacological activity echinochrome A singly and consisting of BAA "Timarin"].

    PubMed

    Artiukov, A A; Popov, A M; Tsybul'skiĭ, A V; Krivoshapko, O N; Poliakova, N V

    2012-01-01

    Pharmacological activity of echinochrome A (EchA) alone and in the biologically active additives (BAA) "Timarin", administered per os has been investigated on volunteers. EchA decreased serum glutatione (GSH) and increased catalase activity 1 h after treatment; catalase activity normalized, while GSH exceeded the initial level 3 h after the treatment. Changes in serum lipid spectrum, demonstrating reduction of the risk atherogenesis were determined.

  11. Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity.

    PubMed

    Brown, Kenneth L; Zou, Xiang; Chen, Guodong; Xia, Zuping; Marques, Helder M

    2004-02-01

    The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are

  12. Amphioxus allantoicase: molecular cloning, expression and enzymatic activity.

    PubMed

    Wang, Yongjun; Zhang, Shicui; Liu, Zhenhui; Li, Hongyan; Wang, Lei

    2005-06-01

    Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.

  13. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

    PubMed

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

    2015-11-10

    AbstractLignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

  14. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

    PubMed

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

    2015-11-01

    Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

  15. Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

    PubMed Central

    Demina, Galina R.; Makarov, Vadim A.; Nikitushkin, Vadim D.; Ryabova, Olga B.; Vostroknutova, Galina N.; Salina, Elena G.; Shleeva, Margarita O.; Goncharenko, Anna V.; Kaprelyants, Arseny S.

    2009-01-01

    Background Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins. PMID:20016836

  16. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    PubMed

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  17. Soil disturbance increases soil microbial enzymatic activity in arid ecoregion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional diversity of the soil microbial community is commonly used in the assessment of soil health as it relates to the activity of soil microflora involved in carbon cycling. Soil microbes in different microenvironments will have varying responses to different substrates, thus catabolic fingerp...

  18. Investigation of the Solubility and Enzymatic Activity of a Thioredoxin-Gelonin Fusion Protein

    DTIC Science & Technology

    1997-05-01

    tosyl-L-lysine chloromethyl ketone TPCK N -tosyl-L-phenylalanine chloromethyl ketone Tris tris( hydroxymethyl )aminomethane trxA gene for thioredoxin UV...synthesis inhibition assay and the RIP- specific N -glycosidase assay. Thioredoxin-gelonin did not exhibit any enzymatic activity in either assay, even at...Inhibition of Purified Gelonin-XOMA ........... 61 Figure 13: UV Photograph of the Polyacrylamide Gel - N -Glycosidase Activity of Purified Gelonin-XOMA

  19. A comparative study on the rectal aminopeptidase enzymatic activities of different species.

    PubMed

    Acartürk, F; Parlatan, Z I

    2003-03-01

    The aim of the present study was to compare the enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A, aminopeptidase B) in rectal homogenates from different species: rabbit, rat, guinea-pig, sheep and human. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity. For this purpose, 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B were employed. The rectal aminopeptidase enzymatic activity was determined spectrofluorometrically. The inhibition of activity of aminopeptidase in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all rectal homogenates. Sheep and guinea-pig had the greatest aminopeptidase activity. The four aminopeptidase activities of rat and rabbit were not significantly different from each other. Human data was not evaluated statistically, due to insufficient sample. But the values of human data was close to those of the rabbit and rat values except for aminopeptidase A. Based on the data of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was rather difficult to determine the aminopeptidase type in the rectal homogenates of the species studied. It has been found that the aminopeptidase activities of rat and rabbit were not statistically different from each other and the human data were close to them.

  20. Influence of Cr(VI) on enzymatic activity of soil

    NASA Astrophysics Data System (ADS)

    Pacha, Jerzy

    1993-03-01

    The inhibitory effect of Cr(VI) on soil hydrolases activity during two different periods (one and six months) was investigated, in order to obtain information on the change in heavy metal toxicity with time. Considering toxicity as the ecological dose-50% (EcD50) toxicity tended to increase over six months for cellulase Cx, protease and acid phosphates and to decrease for amylase. The average EcD50 value varied between 4450 and 1210 ppm for cellulase Cx, 5000 and 2320 ppm for protease, 3830 and 3295 ppm for acid phosphatase, 4030 and over 5000 ppm for amylase.

  1. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    PubMed Central

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, vmax decreased significantly (P < 0.05) and KM increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  2. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

    PubMed

    Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

    2015-12-21

    Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

  3. Microbial enzymatic activities in a pilot-scale MBR experimental plant under different working conditions.

    PubMed

    Molina-Muñoz, M; Poyatos, J M; Rodelas, B; Pozo, C; Manzanera, M; Hontoria, E; Gonzalez-Lopez, J

    2010-01-01

    Phosphatases, glucosidase, protease, esterase and dehydrogenase activities in a MBR (membrane bioreactor) system equipped with ultrafiltration membranes for the treatment of real urban wastewater were measured at different volatile suspended solid (VSS) concentrations, total suspended solid (TSS) concentrations, hydraulic retention times (HRT), temperatures and inflow rates. The results showed the capacity of the MBR system to remove COD and BOD(5) at TSS between 7200 and 13,300 mg/L; HRT values of 8.05 and 15.27 h; inflow rates of 14.67 and 27.81 L/h; and temperatures between 4 and 27 degrees C. The enzymatic activities are influenced by increases in VSS and TSS concentrations. These results suggest that the ability to get adapted to environmental changes of the bacterial populations and their microbial enzymatic activities is essential to understand the biological processes that occur in MBR systems and crucial for proper urban wastewater treatment when using MBR technologies.

  4. Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

    PubMed

    Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

    2015-12-01

    Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc.

  5. Local modulation of steroid action: rapid control of enzymatic activity

    PubMed Central

    Charlier, Thierry D.; Cornil, Charlotte A.; Patte-Mensah, Christine; Meyer, Laurence; Mensah-Nyagan, A. Guy; Balthazart, Jacques

    2015-01-01

    Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17β-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:25852459

  6. Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells.

    PubMed

    Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Gonzalez-Davis, Oscar; Cadena-Nava, Ruben D; Vazquez-Duhalt, Rafael

    2017-04-03

    Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli. The VLPs with enzymatic activity were covered with polyethylene glycol that was functionalized in its distal end with folic acid in order to be recognized by folate receptors exhibited on tumor cells. The capacity of biocatalytic VLPs to be recognized and internalized into tumor cells is demonstrated. The VLP-treated cells showed enhanced capacity for the transformation of the pro-drug tamoxifen, which resulted in an increase of the cell sensitivity to this oncological drug. In this work, the potential use of biocatalytic VLPs vehicles as a delivery system of medical relevant enzymes is clearly demonstrated. In addition to cancer treatment, this technology also offers an interesting platform as nano-bioreactors for intracellular delivery of enzymatic activity for other diseases originated by the lack of enzymatic activity.

  7. Enzymatic activity in the presence of surfactants commonly used in dissolution media, Part 1: Pepsin.

    PubMed

    Guzman, Maria L; Marques, Margareth R; Olivera Me, Maria E; Stippler, Erika S

    2016-01-01

    The United States Pharmacopeia (USP) General Chapters Dissolution 〈711〉 and Disintegration and Dissolution of Dietary Supplements 〈2040〉 allows the use of enzymes in dissolution media when gelatin capsules do not conform to dissolution specifications due to cross linking. Possible interactions between enzymes and surfactants when used together in dissolution media could result in loss of the enzymatic activity. Pepsin is an enzyme commonly used in dissolution media, and in this work, the activity of pepsin was determined in the presence of different surfactants as usually found in case of dissolution tests of certain gelatin capsule formulations. Pepsin enzymatic activity was determined according to the Ninth Edition of the Food Chemicals Codex (FCC) 9 method, in dissolution conditions: simulated gastric fluid, 37 °C and 50 rpm. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), polysorbate 80 (Tween 80) and octoxynol 9 (Triton X100) in concentrations above and below their critical micellar concentrations were selected. Results showed a significant reduction in the activity of pepsin at all the concentrations of SDS assayed. On the contrary, CTAB, Tween 80, and Triton X100 did not alter the enzymatic activity at of pepsin any of the concentration assayed. This data demonstrates a rational selection of the surfactant to be used when pepsin is required in dissolution test.

  8. Enzymatically active biomimetic micropropellers for the penetration of mucin gels

    PubMed Central

    Walker, Debora; Käsdorf, Benjamin T.; Jeong, Hyeon-Ho; Lieleg, Oliver; Fischer, Peer

    2015-01-01

    In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacterium Helicobacter pylori has developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus. PMID:26824056

  9. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  10. Regulation of Nox and Duox Enzymatic Activity and Expression

    PubMed Central

    Lambeth, J. David; Kawahara, Tsukasa; Diebold, Becky

    2007-01-01

    Summary In recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide and other metabolites) are produced in biological systems. Rather than being simply a byproduct of aerobic metabolism, it is now recognized that specific enzymes --- the Nox (NADPH-oxidase) and Duox (Dual oxidase) enzymes ---- seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes. PMID:17602947

  11. Generation of composites for bone tissue-engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means.

    PubMed

    Douglas, Timothy E L; Krawczyk, Grzegorz; Pamula, Elzbieta; Declercq, Heidi A; Schaubroeck, David; Bucko, Miroslaw M; Balcaen, Lieve; Van Der Voort, Pascal; Bliznuk, Vitaliy; van den Vreken, Natasja M F; Dash, Mamoni; Detsch, Rainer; Boccaccini, Aldo R; Vanhaecke, Frank; Cornelissen, Maria; Dubruel, Peter

    2016-11-01

    Mineralization of hydrogels, desirable for bone regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). ALP-loaded gellan gum (GG) hydrogels were mineralized by incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP, MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all values mol/dm(3) , denoted A, B, C, D and E, respectively) were compared. Mineral formation was confirmed by IR and Raman, SEM, ICP-OES, XRD, TEM, SAED, TGA and increases in the the mass fraction of the hydrogel not consisting of water. Ca was incorporated into mineral to a greater extent than Mg in samples mineralized in media A-D. Mg content and amorphicity of mineral formed increased in the order A < B < C < D. Mineral formed in media A and B was calcium-deficient hydroxyapatite (CDHA). Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed in medium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline and amorphous MgP. Young's moduli and storage moduli decreased in dependence of mineralization medium in the order A > B > C > D, but were significantly higher for samples mineralized in medium E. The attachment and vitality of osteoblastic MC3T3-E1 cells were higher on samples mineralized in media B-E (containing Mg) than in those mineralized in medium A (not containing Mg). All samples underwent degradation and supported the adhesion of RAW 264.7 monocytic cells, and samples mineralized in media A and B supported osteoclast-like cell formation. Copyright © 2014 John Wiley & Sons, Ltd.

  12. Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis.

    PubMed

    Carnevali, O; Cionna, C; Tosti, L; Cerdà, J; Gioacchini, G

    2008-01-01

    The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

  13. MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation.

    PubMed

    Peng, Lina; Li, Yan; Xi, Yanping; Li, Wei; Li, Jin; Lv, Ruitu; Zhang, Lei; Zou, Qingping; Dong, Shihua; Luo, Huaibing; Wu, Feizhen; Yu, Wenqiang

    2016-03-01

    Ten-eleven translocation (Tet) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of Tet2 are frequently found in human malignancies, highlighting the essential role of Tet2 in cellular transformation. However, the factors that control Tet enzymatic activity remain largely unknown. Here, we found that methyl-CpG-binding domain protein 3 (MBD3) and its homolog MBD3-like 2 (MBD3L2) can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Moreover, MBD3L2 is more effective than MBD3 in promoting Tet2 enzymatic activity through strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2, and into how that affects Tet2-mediated modulation of its target genes in cancer development. Thus, they have important applications in understanding how dysregulation of Tet2 might contribute to human malignancy.

  14. Serum biochemical profile, enzymatic activity and lipid peroxidation in organs of laying hens fed diets containing cashew nut shell liquid.

    PubMed

    Braz, N M; Freitas, E R; Trevisan, M T S; do Nascimento, G A J; Salles, R P R; Cruz, C E B; Farias, N N P; da Silva, I N G; Watanabe, P H

    2017-03-16

    The objective of this study was to evaluate the effect of feeding laying hens diets containing cashew nut shell liquid (CNSL) as a source of anacardic acid on the blood biochemical parameters as well as the enzymatic activity and lipid peroxidation of liver and tissues of the reproductive system (ovary, magnum, and uterus). A total of 216 Hisex White commercial laying hens were distributed randomly into six treatments, with six replicates of six birds. Treatments consisted of a diet without growth promoter (GP); a diet with GP; and diets without GP, with addition of increasing levels of CNSL (0.25, 0.50, 0.75 and 1.0%). Addition of CNSL to the diet did not affect the blood biochemical parameters (uric acid, creatinine, alanine aminotransferase, aspartate aminotransferase, total cholesterol, high density lipoproteins, low-density lipoproteins and triglycerides), the enzymatic activity (superoxide dismutase and nonprotein sulphydryl groups) in the organs (liver, ovary, magnum and uterus) or the peroxidation of lipids from the blood serum, liver, magnum and uterus (p > 0.05). However, the addition of 0.75% and 1.00% CNSL provided a lower thiobarbituric acid reactive substances content in the birds' ovary (p < 0.001) compared to birds of other treatments, whereas the treatment without the GP provided a higher value. Addition of up to 1% of the CNSL as a source of anacardic acid in the laying hens' diets does not influence blood biochemical parameters or the endogenous enzymatic activity in the liver, ovary, magnum and uterus, but affects the lipid peroxidation in the ovary, although the problem is reduced from the inclusion of 0.75% CNSL.

  15. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    PubMed Central

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-01-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable. PMID:26174478

  16. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  17. Interactive effect of oxytetracycline and lead on soil enzymatic activity and microbial biomass.

    PubMed

    Gao, Minling; Song, Wenhua; Zhou, Qian; Ma, Xiaojun; Chen, Xiaoying

    2013-09-01

    Interactive effect of oxytetracyline (OTC) and lead on soil enzymatic activity and population of microbes was studied in the paper. The results showed effect of pollutants on bacteria, actinomycetes and enzymatic activity increased in the order: (OTC+Pb)>Pb>OTC, (OTC+Pb)>Pb>OTC and (OTC+Pb)>OTC>Pb, respectively. However, impact of pollutants on fungi decreased in the order: (OTC+Pb)

  18. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

  19. Size Dependent Platelet Subpopulations: Relationship of Platelet Volume to Ultrastructure Enzymatic Activity, and Function.

    DTIC Science & Technology

    1983-03-10

    of the present apheresis instruments to separate the larger more functional platelets from the smaller ones. The selective isolation of large... PLATELET VOLUME T. -(U) BOSTON UNIV MA SCHOOL OF I MEDICINE C B THOMPSON ET RL 10 MAR 83 BUSM-93-89 UNIIDN919CA89 /68 6ilfflfllflflflflll l...N00014-79-C-0168 TECHNICAL REPORT NO. 83-08 SIZE DEPENDENT PLATELET SUBPOPULATIONS: RELATIONSHIP OF PLATELET VOLUME TO ULTRASTRUCTURE. ENZYMATIC ACTIVITY

  20. Effect of compost temperature on oxygen uptake rate, specific growth rate and enzymatic activity of microorganisms in dairy cattle manure.

    PubMed

    Miyatake, Fumihito; Iwabuchi, Kazunori

    2006-05-01

    Investigations were carried out to find out the relationship between temperature and microbial activity in dairy cattle manure composting using oxygen uptake rate, specific growth rate and enzymatic activities during autothermal and isothermal composting experiments. In autothermal composting, oxygen uptake rate and specific growth rate were found to be most intensive in order of 43 degrees C, 60 degrees C and 54 degrees C. Isothermal composting at 54 degrees C resulted highest levels of enzymatic activity and promoted the volatile solids reduction. Based on the maximum enzymatic activity, specific growth rate appeared to be more closely linked with microbial activity in compost than with oxygen uptake rate. The enhancement of specific growth rate, enzymatic activity and volatile solids reduction were induced at 54 degrees C in cattle manure composting.

  1. Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.

    PubMed

    Lim, Sung In; Yoon, Sungho; Kim, Yong Hwan; Kwon, Inchan

    2015-04-07

    Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  2. Enzymatically Activated Near Infrared Nanoprobes Based on Amphiphilic Block Copolymers for Optical Detection of Cancer

    PubMed Central

    Ozel, Tugba; White, Sean; Nguyen, Elaine; Moy, Austin; Brenes, Nicholas; Choi, Bernard; Betancourt, Tania

    2015-01-01

    Background and Objective Nanotechnology offers the possibility of creating multi-functional structures that can provide solutions for biomedical problems. The nanoprobes herein described are an example of such structures, where nanoprobes have been designed to provide high specificity and contrast potential for optical detection of cancer. Specifically, enzymatically activated fluorescent nanoprobes (EANPs) were synthesized as cancer-specific contrast agents for optical imaging. Study Design/Materials and Methods EANPs were prepared by nanoprecipitation of blends of poly(lactic acid)-b-poly(ethylene glycol) and poly(lactic-co-glycolic acid)-b-poly(L-lysine). The lysine moieties were then covalently decorated with the near infrared (NIR) fluorescent molecule AlexaFluor-750 (AF750). Close proximity of the fluorescent molecules to each other resulted in fluorescence quenching, which was be reversed by enzymatically mediated cleavage of poly(L-lysine) chains. EANPs were characterized by dynamic light scattering and electron microscopy. Enzymatic development of fluorescence was studied in vitro by fluorescence spectroscopy. Biocompatibility and contrast potential of EANPs were studied in cancerous and noncancerous cells. The potential of the nanoprobes as contrast agents for NIR fluorescence imaging was studied in tissue phantoms. Results Spherical EANPs of ∼100 nm were synthesized via nanoprecipitation of polymer blends. Fluorescence activation of EANPs by treatment with a model protease was demonstrated with up to 15-fold optical signal enhancement within 120 minutes. Studies with MDA-MB-231 breast cancer cells demonstrated the cytocompatibility of EANPs, as well as enhanced fluorescence associated with enzymatic activation. Imaging studies in tissue phantoms confirmed the ability of a simple imaging system based on a laser source and CCD camera to image dilute suspensions of the nanoprobe at depths of up to 4 mm, as well as up to a 13-fold signal

  3. BACE1 and BACE2 Enzymatic Activities in Alzheimer’s Disease

    PubMed Central

    Ahmed, Rachel R.; Holler, Christopher J.; Webb, Robin L.; Li, Feng; Beckett, Tina L.; Murphy, M. Paul

    2009-01-01

    β-Secretase is the rate limiting enzymatic activity in the production of the amyloid-β peptide (Aβ) and is thought to be involved in Alzheimer’s disease (AD) pathogenesis. Although BACE1 (β-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Aβ, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Aβ concentration. These data indicate that BACE1 likely accounts for most of the Aβ produced in the human brain, and that BACE2 activity is not a likely contributor. However, since both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease. PMID:19968762

  4. A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity.

    PubMed

    Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

    2016-01-08

    Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

  5. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  6. The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

    PubMed

    Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

    2013-08-01

    The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries.

  7. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

  8. Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and alfonsino (Beryx splendens) from the Juan Fernandez seamounts area.

    PubMed

    Saavedra, L M; Quiñones, R A; Gonzalez-Saldía, R R; Niklitschek, E J

    2016-06-01

    The aerobic and anaerobic enzymatic activity of two important commercial bathypelagic species living in the Juan Fernández seamounts was analyzed: alfonsino (Beryx splendens) and orange roughy (Hoplostethus atlanticus). These seamounts are influenced by the presence of an oxygen minimum zone (OMZ) located between 160 and 250 m depth. Both species have vertical segregation; alfonsino is able to stay in the OMZ, while orange roughy remains at greater depths. In this study, we compare the aerobic and anaerobic capacity of these species, measuring the activity of key metabolic enzymes in different body tissues (muscle, heart, brain and liver). Alfonsino has higher anaerobic potential in its white muscle due to greater lactate dehydrogenase (LDH) activity (190.2 μmol NADH min(-1) g ww(-1)), which is related to its smaller body size, but it is also a feature shared with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron transport system activities indicate that alfonsino has greater swimming activity level than orange roughy. This species has also a high MDH/LDH ratio in its heart, brain and liver, revealing a potential capacity to conduct aerobic metabolism in these organs under prolonged periods of environmental low oxygen conditions, preventing lactic acid accumulation. With these metabolic characteristics, alfonsino may have increased swimming activity to migrate and also could stay for a period of time in the OMZ. The observed differences between alfonsino and orange roughy with respect to their aerobic and anaerobic enzymatic activity are consistent with their characteristic vertical distributions and feeding behaviors.

  9. Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

    USGS Publications Warehouse

    Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.

    2002-01-01

    Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.

  10. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    SciTech Connect

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

    2016-03-15

    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  11. A new method to determine optimum temperature and activation energies for enzymatic reactions.

    PubMed

    Wojcik, M; Miłek, J

    2016-08-01

    A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.

  12. Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration.

    PubMed

    Fu, Li; Zhang, Fuming; Li, Guoyun; Onishi, Akihiro; Bhaskar, Ujjwal; Sun, Peilong; Linhardt, Robert J

    2014-05-01

    The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.

  13. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices.

    PubMed

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-04-07

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

  14. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

    PubMed Central

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-01-01

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay. PMID:28387379

  15. Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

    PubMed

    Saa, Laura; Grinyte, Ruta; Sánchez-Iglesias, Ana; Liz-Marzán, Luis M; Pavlov, Valeri

    2016-05-04

    The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine.

  16. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  17. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

    PubMed Central

    Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

    2015-01-01

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

  18. Evolution of enzymatic activities and carbon fractions throughout composting of plant waste.

    PubMed

    Jurado, M M; Suárez-Estrella, F; Vargas-García, M C; López, M J; López-González, J A; Moreno, J

    2014-01-15

    Many alternatives for the proper disposal of horticultural plant wastes have been studied, and composting is one of the most attractive due to its insignificant environmental impact and low cost. The quality of compost for agronomical use is related to the degree of organic matter maturation and stabilization. Traditional parameters as well as temperature, ratio C/N, cationic exchange capacity, extractable carbon, or evolution of humificated substances have been successfully used to assess compost maturity and stability. However, microorganisms frequently isolated during composting release a wide range of hydrolytic enzymes, whose activity could apparently give interesting information on the rate of decomposition of organic matter and, therefore, on the product stability. The aim of this work was to study the evolution of some important enzymatic activities during composting of agricultural wastes and their comparison with other chemical parameters commonly employed as quality and maturity indexes, to establish a relationship between the degradation intensity of specific organic carbon fractions throughout the process. In this work, the chemical and biochemical parameters of plant wastes were studied along a composting process of 189 days to evaluate their importance as tools for compost characterization. Results showed an intense enzymatic activity during the first 2-3 weeks of composting (bio-oxidative phase), because of the availability of easily decomposable organic compounds. From a biological point of view, a less intense phase was observed between second and third month of composting (mesophilic or cooling phase). Finally, chemical humification parameters were more closely associated with the period between 119 and 189 days (maturation phase). Significant correlations between the enzymatic activities as well as between enzyme activities and other more traditional parameters were also highlighted, indicating that both kind of indexes can be a reliable tool to

  19. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

    PubMed

    Kalb, Suzanne R; Boyer, Anne E; Barr, John R

    2015-08-31

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

  20. Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner.

    PubMed

    Jenko-Pražnikar, Zala; Petan, Toni; Pungerčar, Jože

    2013-03-01

    Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

  1. Changes in the enzymatic activity of soil samples upon their storage

    NASA Astrophysics Data System (ADS)

    Dadenko, E. V.; Kazeev, K. Sh.; Kolesnikov, S. I.; Val'Kov, V. F.

    2009-12-01

    The influence of the duration and conditions of storage of soil samples on the activity of soil enzymes (catalase, β-fructofuranosidase, and dehydrogenase) was studied for the main soils of southern Russia (different subtypes of chernozems, chestnut soils, brown forest soils, gray forest soils, solonetzes, and solonchaks). The following soil storage conditions were tested: (1) the air-dry state at room temperature, (2) the airdry state at a low positive (in a refrigerator, +4°C) temperature, (3) naturally moist samples at a low positive temperature, and (4) naturally moist samples at a negative (in a freezer, -5°C) temperature. It was found that the sample storing caused significant changes in the enzymatic activities, which depended on the soil type, the land use, the type of enzyme, and the duration and conditions of the sample storage. In the course of the storage, the changes in the enzymatic activity had a nonlinear character. The maximum changes were observed in the initial period (up to 12 weeks). Then, a very gradual decrease in the activity of the studied enzymes was observed. Upon the long-term (>12 weeks) storage under the different conditions, the difference in the activities of the soil enzymes became less pronounced. The storage of soil samples in the air-dried state at room temperature can be recommended for mass investigations.

  2. Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts.

    PubMed

    Hong, Yang-Hee; Jung, Eun Young; Park, Yooheon; Shin, Kwang-Soon; Kim, Tae Young; Yu, Kwang-Won; Chang, Un Jae; Suh, Hyung Joo

    2013-01-01

    This study describes increases in extraction efficiency and the bioconversion of catechins after treatment with several commercial enzymes. Tannase was also used to improve the anti-radical activities of green tea extracts. Enzymatic treatment with various commercial enzymes was introduced to improve the extraction efficiency of polyphenols. The total polyphenol, flavonoid, and catechin contents and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the green tea extract treated with Viscozyme (VG) were significantly higher than those treated with other commercial enzymatic extractions (p<0.05). More than 95% of the epigallocatechingallate (EGCG) and of the epicatechingallate (ECG) was hydrolyzed to epigallocatechin (EGC) and to epicatechin (EC) in successive 20 min treatments with Viscozyme and tannase (TG). Due to its hydrolytic activity, treatment involving tannase resulted in a significant release of gallic acid (GA), EGC, and EC, leading to greater radical scavenging activities. Regarding the IC(50) values of the DPPH and 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, the green tea extract treated with TG showed values of 131.23 and 28.83 µg/mL, VG showed values of 224.70 and 32.54 µg/mL, and normal green tea extract (NG) showed values of 241.11 and 66.27 µg/mL, respectively. These results indicate that successive treatment with Viscozyme and tannase improves the extraction efficiency of polyphenols and increases radical scavenging activities.

  3. Effects of Prochloraz fungicide on soil enzymatic activities and bacterial communities.

    PubMed

    Tejada, Manuel; Gómez, Isidoro; García-Martínez, Ana María; Osta, Paloma; Parrado, Juan

    2011-09-01

    We studied in the laboratory the effect of Prochloraz fungicide on the biological properties (soil enzymatic activities and soil bacterial communities) of a Plaggic Anthrosol. Five hundred grams of soil (<2mm) was mixed with three dosages of Prochloraz (1, 2, and 4 l ha(-1)) for 83 days. A non-Prochloraz polluted soil was used as control. Following commercial recommendations, fungicide was applied four times during the incubation experiment. For all treatments, the soil ergosterol and levels of dehydrogenase, urease, β-glucosidase, and phosphatase activity were measured at nine different times (0, 1, 21, 22, 41, 42, 62, 63, and 83 days). The 16S rDNA-DGGE profiles in all treatments were determined at the beginning and end of the incubation period. At the end of the experiment, a significant decrease in ergosterol by 72.3%, 80.8%, and 83.1%, compared with control soil, was observed when 1, 2, and 4 l ha(-1), respectively, was added. Soil enzymatic activities increased when the Prochloraz applied to the soil increased, possibly because the fungicide is used by bacterial communities as a source of energy and nutrients. The 16S rDNA-DGGE profiles indicated that the fungicide did not negatively affect soil bacterial biodiversity. These results suggested that the fungicide Prochloraz has a very interesting agronomic effect, possibly due to the negative effect on soil fungal population stimulating the growth of soil bacterial activity.

  4. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

    PubMed Central

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C

    2016-01-01

    Summary The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for

  5. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

    PubMed

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C; Wege, Christina

    2016-01-01

    The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for

  6. [Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

    PubMed

    Nersesova, L S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Pogosian, S A; Pogosian, L L; Karalova, E M; Avetisian, A S; Abroian, l O; Karalian, Z A; Akopian, Zh I

    2013-01-01

    The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

  7. Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

    SciTech Connect

    Efrem Mebrahtu, Suzanne Lapi

    2012-12-13

    This application investigated a novel imaging approach to develop methods to incorporate multiple radionuclides into a single peptide at chemoselective sites for simultaneous monitoring of cell-bound protein targets as well as specific enzymatic activity, both of which are associated with enhanced tumor growth and metastasis. This imaging construct was synthesized in such a manner so that the PET radionuclide will remain associated with the tumor cells and the SPECT radionuclide was cleaved from the imaging agent. Measurement of the PET agent only will yield information about the tumor marker density while measurement of the amount of co-localization and mismatch of the two radionuclides will yield information about the enzymatic activity. This coincident measuring technique using both PET and SPECT agents allows us to draw correlations involving the interactions of enzymes (cathepsin, serine-protease urokinase (uPA) and matrix metalloproteases) and other cellular proteins which play a role in cancer growth and metastasis. This technique will allow for studies in xenograft or genetic models of cancer in the same animal at the same time, thus eliminating problems that may occur when trying to invoke comparisons across animals or timepoints. By using radionuclide imaging as opposed to other imaging modalities, this technique has the potential to be translatable and can exploit the high specific activity probes which can be generated with radiotracers. The proof of principle test of this system investigated simultaneous monitoring of matrix metalloprotease (MMP) activity in the extracellular matrix (ECM) as well as density of integrins on the cell surface, both of which can serve as tumor markers. The outcomes/deliverables of this project were as follows: 1. Peptides were synthesized dually labeled at chemospecific sites with PET and SPECT agents. 2. Stability (intrinsic and to radiolysis) and specific activity of these labeled compounds were determined. 3. The

  8. Effect of untreated sewage effluent irrigation on heavy metal content, microbial population and enzymatic activities of soils in Aligarh.

    PubMed

    Bansal, O P; Singh, Gajraj; Katiyar, Pragati

    2014-07-01

    The study pertains to the impact of domestic and industrial sewage water irrigation on the chemical, biological and enzymatic activities in alluvial soils of Aligarh District. Results showed that soil enzymatic [dehydogenase (DHA), acid and alkaline phosphatase, urease and catalase] activities in the soils increased up to 14 days of incubation and thereafter inhibited significantly. The enzymatic activity were in the order sewage effluent > partial sewage effluent > ground water irrigated soils. Increase in soil enzymatic activities up to 2nd week of incubation was due to decomposition of organic matter. Maximum inhibition of enzymatic activities, after 14 days of incubation were found in sewage effluent irrigated soils and minimum in ground water irrigated soils. Similar trend was also seen for microbial population. Soil enzymatic activities and microbial population were significantly and positively correlated with soil organic matter. Results also indicated that the microbial population and enzymatic activities in sewage irrigated soils decreased continually with irrigation period. The average concentration of total heavy metals in sewage irrigated soils and partial sewage irrigated soils increased and was 3 and 2 times higher for Zn; 4.5 and 1.7 times higher for Cu; 3.8 and 2.4 times higher for Cr; 5.7 and 3.5 times higher for Pb; 3.5 and 2.2 times higher for Cd and 2.7 and 2.0 times higher for Ni respectively than that of ground water irrigated soils. Results also showed that though total heavy metals concentration increased with period of sewage irrigation but the concentration of diethylene triamine pentaacetic acid (DTPA) extractable heavy metals in partial sewage irrigated and sewage irrigated soils remained almost same, which might be due to deposition of heavy metals in crops grown on the soils.

  9. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

    PubMed

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

    2013-01-01

    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  10. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains.

  11. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity.

    PubMed

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees

    2015-01-01

    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  12. Integrated catalysis opens new arylation pathways via regiodivergent enzymatic C–H activation

    PubMed Central

    Latham, Jonathan; Henry, Jean-Marc; Sharif, Humera H.; Menon, Binuraj R. K.; Shepherd, Sarah A.; Greaney, Michael F.; Micklefield, Jason

    2016-01-01

    Despite major recent advances in C–H activation, discrimination between two similar, unactivated C–H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant ‘chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C–H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C–H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C–H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps. PMID:27283121

  13. Antibacterial and enzymatic activity of microbial community during wastewater treatment by pilot scale vermifiltration system.

    PubMed

    Arora, Sudipti; Rajpal, Ankur; Bhargava, Renu; Pruthi, Vikas; Bhatia, Akansha; Kazmi, A A

    2014-08-01

    The present study investigated microbial community diversity and antibacterial and enzymatic properties of microorganisms in a pilot-scale vermifiltration system during domestic wastewater treatment. The study included isolation and identification of diverse microbial community by culture-dependent method from a vermifilter (VF) with earthworms and a conventional geofilter (GF) without earthworms. The results of the four months study revealed that presence of earthworms in VF could efficiently remove biochemical oxygen demand (BOD), chemical oxygen demand (COD), total and fecal coliforms, fecal streptococci and other pathogens. Furthermore, the burrowing activity of earthworms promoted the aeration conditions in VF which led to the predominance of the aerobic microorganisms, accounting for complex microbial community diversity. Antibacterial activity of the isolated microorganisms revealed the mechanism behind the removal of pathogens, which is reported for the first time. Specifically, cellulase, amylase and protease activity is responsible for biodegradation and stabilization of organic matter.

  14. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  15. Assessment of cathepsin mRNA expression and enzymatic activity during early embryonic development in the yellowtail kingfish Seriola lalandi.

    PubMed

    Palomino, Jaime; Herrera, Giannina; Torres-Fuentes, Jorge; Dettleff, Phillip; Patel, Alok; Martínez, Víctor

    2017-02-21

    In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.

  16. Assessment of the human fecal microbiota: I. Measurement and reproducibility of selected enzymatic activities

    PubMed Central

    Flores, Roberto; Shi, Jianxin; Gail, Mitchell H.; Ravel, Jacques; Goedert, James J.

    2012-01-01

    Background The intestinal microbial community has major effects on human health, but optimal research methods are unsettled. To facilitate epidemiologic and clinical research, we sought to optimize conditions and to assess reproducibility of selected core functions of the distal gut microbiota, β-glucuronidase and β-glucosidase bioactivities. Methods and results A colorimetric kinetic method was optimized and used to quantify activities of β-glucuronidase and β-glucosidase in human feces. Enzyme detection was optimal with neutral pH, snap freezing in liquid nitrogen, and rapid thawing to 37°C before protein extraction. Enzymatic stability was assessed by delayed freezing for 2–48 hours to mimic field settings. Activities decayed approximately 20% within 2 hours and 40% within 4 hours at room temperature. To formally assess reproducibility, 51 volunteers (25 male; mean age 39) used two devices to self-collect and rapidly chill four replicates of a stool. Devices were compared for mean enzymatic activities and intraclass correlation coefficients (ICC) in paired replicates of the self-collected specimens. Reproducibility was excellent with both devices for β-glucuronidase (ICC 0.92). The larger collection device had significantly higher reproducibility for β-glucosidase (ICC 0.92 vs. 0.76, P<0.0001) and higher mean activities for both enzymes (P<0.0001). Conclusions Optimal measurement of these core activities of the microbiota required a sufficient quantity of rapidly chilled or frozen specimens collected in PBS at pH7.0. Application of these methods to clinical and epidemiologic research could provide insights on how the intestinal microbiota affects human health. PMID:22409163

  17. Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese.

    PubMed

    González, Leticia; Sacristán, Noelia; Arenas, Ricardo; Fresno, José M; Eugenia Tornadijo, M

    2010-08-01

    Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains). Lactococcus strains were those that showed the greatest degree of acidifying and proteolytic activity. The cell-free extracts (CFE) of L. paracasei exhibited the highest level of aminopeptidase activity. The highest level of caseinolytic activity was shown by the CFE of one strain of L. lactis. High values were also obtained with the CFE of Lactobacillus and of several Leuconostoc. The highest level of dipeptidase activity was found amongst the strains of L. lactis. Carboxypeptidase activity was generally very low or undetectable for the majority of strains. The greatest degree of esterolytic activity was detected for Enterococcus.

  18. AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud.

    PubMed

    Rangam, Gopinath; Schmitz, Kerstin-Maike; Cobb, Alexander J A; Petersen-Mahrt, Svend K

    2012-01-01

    Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl > hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

  19. Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases

    PubMed Central

    Tarhonskaya, Hanna; Rydzik, Anna M.; Leung, Ivanhoe K. H.; Loik, Nikita D.; Chan, Mun Chiang; Kawamura, Akane; McCullagh, James S. O.; Claridge, Timothy D. W.; Flashman, Emily; Schofield, Christopher J.

    2014-01-01

    Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest. PMID:24594748

  20. Enzymatic heme oxygenase activity in soluble extracts of the unicellular red alga, Cyanidium caldarium.

    PubMed

    Beale, S I; Cornejo, J

    1984-12-01

    Extracts of the phycocyanin-containing unicellular red alga, Cyanidium caldarium, catalyzed enzymatic cleavage of the heme macrocycle to form the linear tetrapyrrole bilin structure. This is the key first step in the branch of the tetrapyrrole biosynthetic pathway leading to phycobilin photosynthetic accessory pigments. A mixed-function oxidase mechanism, similar to the biliverdin-forming reaction catalyzed by animal cell-derived microsomal heme oxygenase, was indicated by requirements for O2 and a reduced pyridine nucleotide. To avoid enzymatic conversion of the bilin product to phycocyanobilins and subsequent degradation during incubation, mesoheme IX was substituted for the normal physiological substrate, protoheme IX. Mesobiliverdin IX alpha was identified as the primary incubation product by comparative reverse-phase high-pressure liquid chromatography and absorption spectrophotometry. The enzymatic nature of the reaction was indicated by the requirement for cell extract, absence of activity in boiled cell extract, high specificity for NADPH as cosubstrate, formation of the physiologically relevant IX alpha bilin isomer, and over 75% inhibition by 1 microM Sn-protoporphyrin, which has been reported to be a competitive inhibitor of animal microsomal heme oxygenase. On the other hand, coupled oxidation of mesoheme, catalyzed by ascorbate plus pyridine or myoglobin, yielded a mixture of ring-opening mesobiliverdin IX isomers, was not inhibited by Sn-protoporphyrin, and could not use NADPH as the reductant. Unlike the animal microsomal heme oxygenase, the algal reaction appeared to be catalyzed by a soluble enzyme that was not sedimentable by centrifugation for 1 h at 200,000g. Although NADPH was the preferred reductant, small amounts of activity were obtained with NADH or ascorbate. A portion of the activity was retained after gel filtration of the cell extract to remove low-molecular-weight components. Considerable stimulation of activity, particularly in

  1. Vertical and horizontal distributions of microbial abundances and enzymatic activities in propylene-glycol-affected soils.

    PubMed

    Biró, Borbála; Toscano, Giuseppe; Horváth, Nikoletta; Matics, Heléna; Domonkos, Mónika; Scotti, Riccardo; Rao, Maria A; Wejden, Bente; French, Helen K

    2014-01-01

    The natural microbial activity in the unsaturated soil is vital for protecting groundwater in areas where high loads of biodegradable contaminants are supplied to the surface, which usually is the case for airports using aircraft de-icing fluids (ADF) in the cold season. Horizontal and vertical distributions of microbial abundance were assessed along the western runway of Oslo Airport (Gardermoen, Norway) to monitor the effect of ADF dispersion with special reference to the component with the highest chemical oxygen demand (COD), propylene glycol (PG). Microbial abundance was evaluated by several biondicators: colony-forming units (CFU) of some physiological groups (aerobic and anaerobic heterotrophs and microscopic fungi), most probable numbers (MPN) of PG degraders, selected catabolic enzymatic activities (fluorescein diacetate (FDA) hydrolase, dehydrogenase, and β-glucosidase). High correlations were found between the enzymatic activities and microbial counts in vertical soil profiles. All microbial abundance indicators showed a steep drop in the first meter of soil depth. The vertical distribution of microbial abundance can be correlated by a decreasing exponential function of depth. The horizontal trend of microbial abundance (evaluated as total aerobic CFU, MPN of PG-degraders, and FDA hydrolase activity) assessed in the surface soil at an increasing distance from the runway is correlated negatively with the PG and COD loads, suggesting the relevance of other chemicals in the modulation of microbial growth. The possible role of potassium formate, component of runway de-icers, has been tested in the laboratory by using mixed cultures of Pseudomonas spp., obtained by enrichment with a selective PG medium from soil samples taken at the most contaminated area near the runway. The inhibitory effect of formate on the growth of PG degraders is proven by the reduction of biomass yield on PG in the presence of formate.

  2. Enzymatic and metabolic activities of four anaerobic sludges and their impact on methane production from ensiled sorghum forage.

    PubMed

    Sambusiti, C; Rollini, M; Ficara, E; Musatti, A; Manzoni, M; Malpei, F

    2014-03-01

    Biochemical methane potential (BMP) tests were run on ensiled sorghum forage using four inocula (urban, agricultural, mixture of agricultural and urban, granular) and differences on their metabolic and enzymatic activities were also discussed. Results indicate that no significant differences were observed in terms of BMP values (258±14NmLCH4g(-1)VS) with a slightly higher value when agricultural sludge was used as inoculum. Significant differences can be observed among different inocula, in terms of methane production rate. In particular the fastest biomethanization occurred when using the urban sludge (hydrolytic kinetic constant kh=0.146d(-1)) while the slowest one was obtained from the agricultural sludge (kh=0.049d(-1)). Interestingly, positive correlations between the overall enzymatic activities and methane production rates were observed for all sludges, showing that a high enzymatic activity may favour the hydrolysis of complex substrate and accelerate the methanization process of sorghum.

  3. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT PROGRAMS Consistency of Federal Activities Having Interstate Coastal Effects § 930.154 Listing activities... listed activity will occur, as well as with relevant Federal agencies. (c) Demonstrate effects....

  4. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT PROGRAMS Consistency of Federal Activities Having Interstate Coastal Effects § 930.154 Listing activities... listed activity will occur, as well as with relevant Federal agencies. (c) Demonstrate effects....

  5. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    PubMed

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

  6. Correlation between quantified promoter methylation and enzymatic activity of O6-methylguanine-DNA methyltransferase in glioblastomas.

    PubMed

    Kishida, Yugo; Natsume, Atsushi; Toda, Hiroshi; Toi, Yuki; Motomura, Kazuya; Koyama, Hiroko; Matsuda, Keiji; Nakayama, Osamu; Sato, Makoto; Suzuki, Masaaki; Kondo, Yutaka; Wakabayashi, Toshihiko

    2012-04-01

    The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT, AGT) is a determinant of the resistance of tumor cells to alkylating anticancer agents that target the O(6) position of guanine. MGMT promoter methylation in tumors is regarded as the most common predictor of the responsiveness of glioblastoma to alkylating agents. However, MGMT promoter methylation status has been investigated mainly by methylation-specific PCR, which is a qualitative and subjective assay. In addition, the actual enzymatic activities associated with the methylation status of MGMT have not been explored. In the present study, MGMT promoter methylation in glioblastomas was quantified by bisulfite pyrosequencing, and its correlation with enzymatic activity was determined using a novel quantitative assay for studying the functional activity of MGMT. MGMT enzymatic activity was assessed using fluorometrically labeled oligonucleotide substrates containing MGMT-specific DNA lesions and capillary electrophoresis to detect and quantify these lesions. In comparison with existing traditional assays, this assay was equally sensitive but less time consuming and easier to perform. MGMT promoter methylation was assessed in 41 glioblastomas by bisulfite pyrosequencing, and five samples with different values were chosen for comparison with enzymatic assays. Bisulfite pyrosequencing using primers designed to work in the upstream promoter regions of MGMT demonstrated high quantitative capability and reproducibility in triplicate measurements. In comparative studies, MGMT promoter methylation values obtained by bisulfite pyrosequencing were inversely proportional to the measured enzymatic activity. The present results indicate that the quantification of MGMT methylation by bisulfite pyrosequencing represents its enzymatic activity and thus, its therapeutic responsiveness to alkylating agents.

  7. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    PubMed Central

    Vaz, Aline B. M.; Rosa, Luiz H.; Vieira, Mariana L. A.; de Garcia, Virginia; Brandão, Luciana R.; Teixeira, Lia C. R. S.; Moliné, Martin; Libkind, Diego; van Broock, Maria; Rosa, Carlos A.

    2011-01-01

    The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates. PMID:24031709

  8. Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

    PubMed

    Kuwahara, Kenrick; Fang, Josephine Y; Yang, Zhi; Han, Bo

    2011-12-01

    Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.

  9. Many disease-associated variants of hTERT retain high telomerase enzymatic activity

    PubMed Central

    Zaug, Arthur J.; Crary, Sharon M.; Jesse Fioravanti, Matthew; Campbell, Kristina; Cech, Thomas R.

    2013-01-01

    Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays. PMID:23901009

  10. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  11. Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill.

    PubMed

    Hakime-Silva, Ricardo A; Vellosa, José C R; Khalil, Najeh M; Khalil, Omar A K; Brunetti, Iguatemy L; Oliveira, Olga M M F

    2013-09-01

    Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• -), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

  12. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  13. Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

    PubMed

    Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

    2016-03-01

    The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian.

  14. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts.

    PubMed

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  15. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts

    PubMed Central

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0–3 mm, transitional zone (TZ) 3–7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98–0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86–113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  16. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway.

    PubMed

    Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

  17. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

    PubMed Central

    Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

  18. A novel technical approach for the measurement of individual ACAT-1 and ACAT-2 enzymatic activity in the testis.

    PubMed

    Chen, Li; Lafond, Julie; Pelletier, R-Marc

    2009-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC(50) values of 100 and 1,000 microM, respectively, in the tubules. Next, the inhibitor concentration (100 microM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 microg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 microg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0-1,000 microM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.

  19. Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase.

    PubMed

    Gonzalez, M A; Cooperman, B S

    1986-11-04

    Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].

  20. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

    PubMed

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

    2016-02-01

    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

  1. Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase.

    PubMed

    Wang, Chunxing; Zhang, Huiling; Ren, Dajun; Li, Qian; Zhang, Shuqin; Feng, Tao

    2015-09-01

    This work investigates the effect of direct-current electric field on the extracellular enzymatic activity, concentration and other experimental parameters of laccase from Trametes versicolor. The results showed that laccase could significantly contribute to the change of pH at the end of graphite electrode. In addition, it increased the electrical conductivity of the water. In the experiment, the optimum pH and catalytic pH range for laccase activity were 3.0 and pH 2.5-4.0. The application of 6 V direct current showed significant effects on the laccase enzyme activity. The activity of laccase was enhanced in the anodic region, but at the same time was strongly inhibited at the cathode. The electric charge characteristics of laccase were changed when exposed to electric field, and some laccases molecules moved to the anode, which produced a slight migration phenomenon. This study is the basis of combination of laccase and electrical technology, at the same time, providing a new direction of enhancing laccase activity. Compared to immobilization, using electric field is simple, no chemical additives, and great potential.

  2. 18- and 24-month-olds' discrimination of gender-consistent and inconsistent activities.

    PubMed

    Hill, Sara E; Flom, Ross

    2007-02-01

    18- and 24-month-olds' ability to discriminate gender-stereotyped activities was assessed. Using a preferential looking paradigm, toddlers viewed male and female actors performing masculine and feminine-stereotyped activities. Consistent with our predictions, and previous research, 24-month-olds, but not 18-month-olds, looked longer at the gender-inconsistent activities than the gender-consistent activities. Results are discussed in terms of toddlers emerging gender stereotypes and perception of everyday events.

  3. A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation.

    PubMed

    Mata, Graça; do Rosário, Virgílio E; Iley, Jim; Constantino, Luís; Moreira, Rui

    2012-01-15

    O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

  4. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

    PubMed

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

    2016-11-14

    the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

  5. Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments

    SciTech Connect

    Meyer-Reil, L.

    1987-08-01

    Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates; incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events.

  6. Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

    PubMed

    Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

    2009-08-12

    The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

  7. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    PubMed Central

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  8. An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation

    PubMed Central

    Young, Neal S.; Levin, Jack; Prendergast, Robert A.

    1972-01-01

    Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate. Images PMID:4624351

  9. Toxicological effects of dimethomorph on soil enzymatic activity and soil earthworm (Eisenia fetida).

    PubMed

    Wang, Caixia; Zhang, Qingming; Wang, Feifei; Liang, Wenxing

    2017-02-01

    The objective of this study was to evaluate the toxicity of the fungicide dimethomorph to soil microbial activity and the earthworm Eisenia fetida. Multiple biomarkers, namely, four soil enzymes (urease, dehydrogenase, invertase, and acid phosphatase), four earthworm biochemical indices (dismutase, catalase, cellulase, and malondialdehyde), and the transcriptional levels of both target genes (dismutase and catalase) were measured at 1, 10, and 100 mg kg(-1) after 1, 7, 21, and 28 days. The degradation rate of dimethomorph in soil was also determined, and the results indicated that most parameters did not differ from the controls at 1 and 10 mg kg(-1) dimethomorph by the last exposure time (28 d). However, high concentrations (100 mg kg(-1)) of dimethomorph had varying effects on soil enzymatic activity and earthworms. These effects gradually decreased with prolonged exposure times. Positive correlations (R(2) > 0.57) between the target gene expression levels and antioxidant enzyme activities were observed in this study. We also found that earthworms have improved soil microbial activity and accelerated the degradation of dimethomorph. Overall, higher concentrations of dimethomorph might pose an ecological hazard to soil environments in the short term.

  10. Cell-free extracellular enzymatic activity is linked to seasonal temperature changes: a case study in the Baltic Sea

    NASA Astrophysics Data System (ADS)

    Baltar, Federico; Legrand, Catherine; Pinhassi, Jarone

    2016-05-01

    Extracellular enzymatic activities (EEAs) are a crucial step in the degradation of organic matter. Dissolved (cell-free) extracellular enzymes in seawater can make up a significant contribution of the bulk EEA. However, the factors controlling the proportion of dissolved EEA in the marine environment remain unknown. Here we studied the seasonal changes in the proportion of dissolved relative to total EEA (of alkaline phosphatase (APase), β-glucosidase (BGase), and leucine aminopeptidase (LAPase)), in the Baltic Sea for 18 months. The proportion of dissolved EEA ranged between 37 and 100, 0 and 100, and 34 and 100 % for APase, BGase, and LAPase, respectively. A consistent seasonal pattern in the proportion of dissolved EEA was found among all the studied enzymes, with values up to 100 % during winter and < 40 % during summer. A significant negative relation was found between the proportion of dissolved EEA and temperature, indicating that temperature might be a critical factor controlling the proportion of dissolved relative to total EEA in marine environments. Our results suggest a strong decoupling of hydrolysis rates from microbial dynamics in cold waters. This implies that under cold conditions, cell-free enzymes can contribute to substrate availability at large distances from the producing cell, increasing the dissociation between the hydrolysis of organic compounds and the actual microbes producing the enzymes. This might also suggest a potential effect of global warming on the hydrolysis of organic matter via a reduction of the contribution of cell-free enzymes to the bulk hydrolytic activity.

  11. Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase.

    PubMed

    Sepasi Tehrani, H; Moosavi-Movahedi, A A; Ghourchian, H; Ahmad, F; Kiany, A; Atri, M S; Ariaeenejad, Sh; Kavousi, K; Saboury, A A

    2013-12-01

    Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.

  12. Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass.

    PubMed

    Kang, Dae Hee; Hyeon, Jeong Eun; You, Seung Kyou; Kim, Seung Wook; Han, Sung Ok

    2014-12-20

    Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

  13. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    PubMed

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  14. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  15. Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

    SciTech Connect

    Wheatly, M.A.; Phillips, C.R.

    1984-06-01

    Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-line of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.

  16. Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development.

    PubMed

    Cesarino, Igor; Araújo, Pedro; Sampaio Mayer, Juliana Lischka; Paes Leme, Adriana Franco; Mazzafera, Paulo

    2012-06-01

    Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

  17. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata).

    PubMed

    Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong

    2014-11-26

    An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical.

  18. Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

    PubMed

    Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Lejbkowicz, Flavio; Rennert, Hedy S; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

    2015-09-01

    The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

  19. Evidence of sub-vent biosphere: enzymatic activities in 308 °C deep-sea hydrothermal systems at Suiyo seamount, Izu Bonin Arc, Western Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Takano, Yoshinori; Edazawa, Yae; Kobayashi, Kensei; Urabe, Tetsuro; Marumo, Katsumi

    2005-01-01

    A high-temperature deep-sea hydrothermal system related to dacitic arc-volcanism was drilled using a tethered, submarine rock-drill system as a part of the Archaean Park Project. The benthic multi-coring system (BMS) employed allowed for direct sampling of microorganisms, rocks and fluids beneath hydrothermal vents. The samples examined in this study were from sites APSK 05 and APSK 07 on the Suiyo Seamount of the Izu-Bonin Arc in the Pacific Ocean. Based on the vertical distribution of samples derived from this vigorous sub-vent environment, a model of deep-sea subterranean chemistry and biology was determined detailing optimal microbial activities. Deep-sea hydrothermal sub-vent core samples of dacitic arc-volcanism obtained at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean were analyzed for acid and alkaline phosphatase enzymatic activities. Useful biomarkers of acid phosphatase (ACP) and alkaline phosphatase (ALP) enzymatic activities were positively correlated against each other and was greatest at the partial middle core sequences; ACP and ALP activities determined were as high as 5.10 and 6.80 nmol/min/g rock, respectively. Biochemical indicators of ACP and ALP were consistent with the origin of biogenic amino acids occupied in the sub-vent region and microbial cell number in the fluid. The significant enzymatic activities demonstrated in this study provides crucial evidence that sub-vent regions represent part of the previously unknown extreme-environment biosphere, extending the known subterranean habitable spaces of, for example, extremophilic microbes. This boring trial was first example of discharging high temperature hydrothermal activities at the frontal arc volcanoes.

  20. Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

    PubMed

    Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

    2016-11-01

    A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect.

  1. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  2. Formation of marine snow and enhanced enzymatic activities in oil-contaminated seawater

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; McKay, L.; Yang, T.; Rhodes, B.; Nigro, L.; Gutierrez, T.; Teske, A.; Arnosti, C.

    2010-12-01

    The fate of oil spilled into the ocean depends on its composition, as well as on biological, chemical, and physical characteristics of the spill site. We investigated the effects of oil addition from the Deepwater Horizon (DH) spill on otherwise uncontaminated water collected close to the spill site. Incubation on a roller table mimicked the physical dynamics of natural seawater, leading to the formation of marine snow-oil aggregates. We measured the enzymatic activities of heterotrophic microbes associated with the aggregates and in the surrounding water, and assessed microbial population and community composition as oil-marine snow aggregates formed and aged in the water. Surface seawater taken near the spill site in May 2010 that had no visible crude oil was incubated in 1-l glass bottles with (oil-bottles) and without (no-oil bottles) a seawater-oil mixture collected from the same site. In the oil-bottles formation of brownish, densely packed marine snow (2-3 cm diameter) was observed within the first hour of the roller table incubation. In contrast no-oil bottles showed aggregate formation only after 3 days, and aggregates were almost transparent, less abundant, and smaller in size (< 1cm diameter). Subsamples of the water surrounding the aggregates were taken throughout 21 days of the roller table incubation, and analyzed for bacterial abundance and community structure as well as the activities of hydrolytic enzymes that are used by heterotrophic bacteria to degrade organic matter. We monitored oil-degrading activities with MUF-stearate and -butyrate, and also measured b-glucosidase, alkaline phosphatase, aminopeptidase, and six different polysaccharide hydrolase activities. Enzymatic activities were up to one order of magnitude higher in the oil-bottles compared with the no-oil bottles throughout the entire incubation time. Butyrate hydrolysis was elevated throughout the time course of the incubation, and stearate hydrolysis was particularly high over the

  3. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  4. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    PubMed

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-11-20

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  5. Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.

    PubMed

    Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui

    2014-10-01

    The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates.

  6. Green Tea and Bone Marrow Transplantation: From Antioxidant Activity to Enzymatic and Multidrug-resistance Modulation.

    PubMed

    Peluso, Ilaria; Palmery, Maura; Vitalone, Annabella

    2016-10-25

    Epigallocatechin-3-gallate (EGCG), the main flavonoid of green tea (GT), could play an active role in the prevention of oxidative-stress-related diseases, such as hematologic malignancies. Some effects of EGCG are not imputable to antioxidant activity, but involve modulation of antioxidant enzymes and uric acid (UA) levels. The latter is the major factor responsible of the plasma non-enzymatic antioxidant capacity (NEAC). However, hyperuricemia is a frequent clinical feature caused by tumor lysis syndrome or cyclosporine side effects, both before and after bone marrow transplantation (BMT). Besides this, food-drug interactions could be associated with GT consumption and could have clinical implications. The molecular mechanisms involved in the redox and drug metabolizing/transporting pathways were discussed, with particular reference to the potential role of GT and EGCG in BMT. Moreover, on reviewing data on NEAC, isoprostanes, uric acid, and various enzymes from human studies on GT, its extract, or EGCG, an increase in NEAC, without effect on isoprostanes, and contrasting results on UA and enzymes were observed. Currently, few and contrasting available evidences suggest caution for GT consumption in BMT patients and more studies are needed to better understand the potential impact of EGCG on oxidative stress and metabolizing/transporting systems.

  7. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    PubMed Central

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  8. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine.

    PubMed

    Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G

    2014-01-01

    In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future.

  9. Inhibition of the enzymatic activity of heme oxygenases by azole-based antifungal drugs.

    PubMed

    Kinobe, Robert T; Dercho, Ryan A; Vlahakis, Jason Z; Brien, James F; Szarek, Walter A; Nakatsu, Kanji

    2006-10-01

    Ketoconazole (KTZ) and other azole antifungal agents are known to have a variety of actions beyond the inhibition of sterol synthesis in fungi. These drugs share structural features with a series of novel heme oxygenase (HO) inhibitors designed in our laboratory. Accordingly, we hypothesized that therapeutically used azole-based antifungal drugs are effective HO inhibitors. Using gas chromatography to quantify carbon monoxide formation in vitro and in vivo, we have shown that azole-containing antifungal drugs are potent HO inhibitors. Terconazole, sulconazole, and KTZ were the most potent drugs with IC(50) values of 0.41 +/- 0.01, 1.1 +/- 0.4, and 0.3 +/- 0.1 microM for rat spleen microsomal HO activity, respectively. Kinetic characterization revealed that KTZ was a noncompetitive HO inhibitor. In the presence of KTZ (2.5 and 10 microM), K(m) values for both rat spleen and brain microsomal HO were not altered; however, a significant decrease in the catalytic capacity (V(max)) was observed (P < 0.005). KTZ was also found to weakly inhibit nitric-oxide synthase with an IC(50) of 177 +/- 2 microM but had no effect on the enzymatic activity of NADPH cytochrome P450 reductase. Because these drugs were effective within the concentration range observed in humans, it is possible that inhibition of HO may play a role in some of the pharmacological actions of these antimycotic drugs.

  10. Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

    NASA Astrophysics Data System (ADS)

    Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

    2005-04-01

    A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

  11. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance

    PubMed Central

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-01-01

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities. PMID:26584633

  12. Immobilization of α-amylase onto a calix[4]arene derivative: Evaluation of its enzymatic activity.

    PubMed

    Veesar, Irshad Ali; Solangi, Imam Bakhsh; Memon, Shahabuddin

    2015-06-01

    In order to enhance the cost-effectiveness practicability of enzymes in many industries such as pharmaceutical, food, medical and some other technological processes, there is great need to immobilize them onto a solid supports. In this study, a new and efficient immobilization of α-amylase from Saccharomyces cerevisiae has been developed by using the surface functionalization of calix[4]arene as support. A glutaraldehyde-containing amino group functionalized calix[4]arene was used to immobilize α-amylase covalently. In this procedure, imide bonds are formed between amino groups on the protein and aldehyde groups on the calix[4]arene surface. The surface modified support was characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM). The effect of various preparation conditions on the immobilized α-amylase process such as immobilization time, enzyme concentration, temperature and pH were investigated. The influence of pH and temperature on the activity of free and immobilized α-amylase was also studied using starch as substrate. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized α-amylase were 25°C and 7, respectively. Compared to the free enzyme, the immobilized α-amylase retained 85% of its original activity and exhibited significant thermal stability than the free one and excellent durability.

  13. Post-Effort Changes in Activity of Traditional Diagnostic Enzymatic Markers in Football Players’ Blood

    PubMed Central

    Chamera, Tomasz; Spieszny, Michał; Klocek, Tomasz; Kostrzewa-Nowak, Dorota; Nowak, Robert; Lachowicz, Milena; Buryta, Rafał; Ficek, Krzysztof; Eider, Jerzy; Moska, Waldemar; Cięszczyk, Paweł

    2015-01-01

    Summary Background Long-term and intensive physical effort causes metabolic and biochemical adaptations for both athletic and non-athletic objectives. Knowing the importance of aerobic training in football players, the aim of this study was to evaluate changes in the activity of: creatinine kinase (CK), creatine kinase MB (CKMB), lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (HBDH), cholinesterase (ChE) and alkaline phosphatase (ALP) in response to a semi-long distance outdoor run under aerobic conditions among both female and male football players. Methods Sixteen participants aged 21.9±2 years (women) and 18.4±0.5 years (men), all of them voluntarily recruited football players, took part in an outdoor run, the women covering a distance of 7.4±0.3 km while men covered a distance of 10.7±1.0 km. Plasma activities of the studied enzymes were determined using an appropriate diagnostic assay kit. Results Our results indicate that total LDH activity could be a useful tool in evaluating physical fitness among athletes. We simultaneously established that ChE could not be a marker useful in assessing metabolic response to physical effort in athletes. Moreover, our results suggest that post-effort changes in ALP activity might be used to estimate early symptoms of certain vitamin deficiencies in an athlete’s diet. Conclusions We confirmed that the assessment of activity of selected traditional diagnostic enzymatic markers provides information about muscle state after physical effort. PMID:28356830

  14. Molecular docking studies and anti-enzymatic activities of Thai mango seed kernel extract against snake venoms.

    PubMed

    Leanpolchareanchai, Jiraporn; Pithayanukul, Pimolpan; Bavovada, Rapepol; Saparpakorn, Patchreenart

    2009-03-31

    The ethanolic extract from seed kernels of Thai mango (MSKE) (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloyl glucopyranose) exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A(2) (PLA(2)), hyaluronidase and L-amino acid oxidase (LAAO) of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK)venoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly supported by in vivo tests. Molecular docking studies indicated that the phenolic molecules of the MSKE could selectively bind to the active sites or their proximity, or modify conserved residues that are critical for the catalysis of PLA(2), and selectively bind to the LAAO binding pocket of both CR and NK venoms and thereby inhibit their enzymatic activities. The results imply a potential use of MSKE against snake venoms.

  15. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

    PubMed

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A

    1981-10-25

    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

  16. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells

    PubMed Central

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B.; Chow, K. Martin; Rodgers, David W.; Quintanilla-Vega, Betzabet

    2015-01-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer’s disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1–20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms. PMID:25673500

  17. Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells.

    PubMed

    Chin-Chan, Miguel; Segovia, José; Quintanar, Liliana; Arcos-López, Trinidad; Hersh, Louis B; Chow, K Martin; Rodgers, David W; Quintanilla-Vega, Betzabet

    2015-05-01

    Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1-20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms.

  18. Enzymatic Activity of the Mycelium Compared with Oospore Development During Infection of Pea Roots by Aphanomyces euteiches.

    PubMed

    Kjøller, R; Rosendahl, S

    1998-09-01

    ABSTRACT To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alkaline phosphatase activity in the roots. Additionally, enzyme activity was measured after electrophoresis of an A. euteiches-specific glucose-6-phosphate isozyme. Development of oospores in the roots was measured after staining the oospores with trypan blue. In plants inoculated with the higher zoospore concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died. In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.

  19. Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.

    PubMed

    Basu, Swarna; Campagnola, Paul J

    2004-01-01

    We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 10(5)-10(6) M(-1) s(-1)and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix.

  20. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    PubMed Central

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

  1. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell.

    PubMed

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.

  2. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    PubMed Central

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  3. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  4. Changes in antioxidant and antiinflammatory activity of black bean (Phaseolus vulgaris L.) protein isolates due to germination and enzymatic digestion.

    PubMed

    López-Barrios, Lidia; Antunes-Ricardo, Marilena; Gutiérrez-Uribe, Janet A

    2016-07-15

    Germination is an inexpensive process to improve the nutritional properties of legumes. The effect of germinating black bean seeds on the production of cotyledon protein hydrolysates (CPH) with antioxidant and antiinflammatory activities was analyzed in this research. After simulated enzymatic digestion, the oxygen radical absorbance capacity (ORAC) of CPH obtained from germinated black beans was lower than that observed for raw cotyledons. There were no significant differences among CPH cellular antioxidant activities (CAA), except for the high CAA of the 120 min hydrolysate obtained from one day germinated black bean cotyledons. The most significant changes due to germination and enzymatic hydrolysis were observed for the inhibition of nitric oxide (NO) production in macrophages. The NO synthesis inhibition observed for raw CPH was reduced after simulated gastrointestinal digestion but for germinated samples the inhibition was doubled. Peptides derived from cell wall proteins produced during germination could be responsible of antiinflammatory activity.

  5. Mitomycin C-DNA adducts generated by DT-diaphorase. Revised mechanism of the enzymatic reductive activation of mitomycin C.

    PubMed

    Suresh Kumar, G; Lipman, R; Cummings, J; Tomasz, M

    1997-11-18

    Mitomycin C (MC) was reductively activated by DT-diaphorase [DTD; NAD(P)H:quinone oxidoreductase] from rat liver carcinoma cells in the presence of Micrococcus lysodeicticus DNA at pH 5.8 and 7.4. The resulting alkylated MC-DNA complexes were digested to the nucleoside level and the covalent MC-nucleoside adducts were separated, identified, and quantitatively analyzed by HPLC. In analogous experiments, two other flavoreductases, NADH-cytochrome c reductase and NADPH-cytochrome c reductase, as well as two chemical reductive activating agents Na2S2O4 and H2/PtO2 were employed as activators for the alkylation of DNA by MC. DTD as well as all the other activators generated the four known major guanine-N2-MC adducts at both pHs. In addition, at the lower pH, the guanine-N7-linked adducts of 2,7-diaminomitosene were detectable in the adduct patterns. At a given pH all the enzymatic and chemical reducing agents generated very similar adduct patterns which, however, differed dramatically at the acidic as compared to the neutral pH. Overall yield of MC adducts was 3-4-fold greater at pH 7.4 than at 5. 8 except in the case of DTD when it was 4-fold lower. Without exception, however, cross-link adduct yields were greater at the acidic pH (2-10-fold within the series). The ratio of adducts of bifunctional activation to those of monofunctional activation was 6-20-fold higher at the acidic as compared to the neutral pH. A comprehensive mechanism of the alkylation of DNA by activated MC was derived from the DNA adduct analysis which complements earlier model studies of the activation of MC. The mechanism consists of three competing activation pathways yielding three different DNA-reactive electrophiles 11, 12, and 17 which generate three unique sets of DNA adducts as endproducts. The relative amounts of these adducts are diagnostic of the relative rates of the competing pathways in vitro, and most likely, in vivo. Factors that influence the relative rates of individual pathways

  6. Activation and stabilization of the hydroperoxide lyase enzymatic extract from mint leaves (Mentha spicata) using selected chemical additives.

    PubMed

    Akacha, Najla B; Karboune, Salwa; Gargouri, Mohamed; Kermasha, Selim

    2010-03-01

    The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t (1/2)), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at -20 degrees Celsius and 4 degrees Celsius. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive.

  7. Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

    PubMed

    Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

    2015-10-01

    Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

  8. [Effect of Siwu decoction and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats].

    PubMed

    Liang, Miao; Ma, Zeng-Chun; Yi, Jian-Feng; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Tang, Xiang-Lin; Li, Hua; Shen, Guo-Lin; Gao, Yue

    2013-11-01

    To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.

  9. Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

    PubMed

    Vincenzi, Simone; Tolin, Serena; Cocolin, Luca; Rantsiou, Kalliopi; Curioni, Andrea; Rolle, Luca

    2012-06-30

    During the off-vine natural withering process of Erbaluce (white) grapes to obtain "Erbaluce Caluso" Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as "green" (L*=40.3, a*=-0.56, b*=15.20), "gold" (L*=37.7, a*=5.01, b*=14.12) and "blue" (L*=28.6, a*=0.89, b*=-0.67). The three groups of berries had different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the samples. Compared to the green and

  10. Epigenetic Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue-Specific and Global Inhibition of EZH2 Enzymatic Activities

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-14-1-0232 TITLE: Epigenetic Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue - Specific and Global...Therapy of Hematopoietic Malignancies: Novel Approaches for Tissue - Specific and Global Inhibition of EZH2 Enzymatic Activities 5b. GRANT NUMBER...binding of EZH2 in B- versus T- cell lineages, and to identify the responsible tissue -specific recruiters. 2. KEYWORDS: Hematopoietic cancer, PRC2

  11. KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity.

    PubMed

    Midorikawa, Ryosuke; Takei, Yosuke; Hirokawa, Nobutaka

    2006-04-21

    In brain development, apoptosis is a physiological process that controls the final numbers of neurons. Here, we report that the activity-dependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (KIF4), a microtubule-based molecular motor. The C-terminal domain of KIF4 is a module that suppresses the activity of poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme known to maintain cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, calcium signaling mediated by CaMKII induces dissociation of KIF4 from PARP-1, resulting in upregulation of PARP-1 activity, which supports neuron survival. After dissociation from PARP-1, KIF4 enters into the cytoplasm from the nucleus and moves to the distal part of neurites in a microtubule-dependent manner. We suggested that KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development.

  12. Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage.

    PubMed

    Cervellati, Carlo; Sticozzi, Claudia; Romani, Arianna; Belmonte, Giuseppe; De Rasmo, Domenico; Signorile, Anna; Cervellati, Franco; Milanese, Chiara; Mastroberardino, Pier Giorgio; Pecorelli, Alessandra; Savelli, Vinno; Forman, Henry J; Hayek, Joussef; Valacchi, Giuseppe

    2015-10-01

    A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

  13. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    PubMed Central

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  14. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

    PubMed

    Rouault, Morgane; Rash, Lachlan D; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2006-05-09

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

  15. Effects of partial decerebration and hypophyseal allograft in the thymus of chicken embryos: thymostimulin localization and enzymatic activities.

    PubMed

    Aita, M; Romano, N

    2006-01-01

    Changes in chicken embryo thymus after partial decerebration (including the hypophysis) and hypophyseal allograft were investigated. Chicken embryos were partially decerebrated at 36-40 hr of incubation and on day 12 received a hypophyseal allograft from 18-day-old donor embryos. The embryonic thymuses were collected on day 18 and examined with histological methods, tested for the anti-thymostimulin-like immune-reaction, and for histoenzymatic activities and compared with normal and sham-operated embryos at the same age. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-thymostimulin, succinic dehydrogenase and ATPase enzymatic activities tested, yielded negative reactions. In partially decerebrated hypophyseal allografted embryos, the same thymic compartments improved and anti-thymostimulin-like immune-reaction and enzymatic activities partially recovered. These findings confirmed the key role of hypophysis in thymic ontogenic development and provided new information in metabolic enzymatic pathways and synthesis of a thymostimulin-like substance in the thymus.

  16. Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity

    SciTech Connect

    Bergh, M.L.E.; Cepko, C.L.; Wolf, D.; Robbins, P.W.

    1987-06-01

    Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors the authors studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37/sup 0/C. At 26/sup 0/C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

  17. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii.

    PubMed

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  18. Enzymatic activities of allergen extracts from three species of dust mites and cockroaches commonly found in Korean home.

    PubMed

    Jeong, Kyoung Yong; Kim, Chungryul; Yong, Tai-Soon

    2010-06-01

    Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.

  19. Influence of short-time imidacloprid and acetamiprid application on soil microbial metabolic activity and enzymatic activity.

    PubMed

    Wang, Fei; Yao, Jun; Chen, Huilun; Yi, Zhengji; Choi, Martin M F

    2014-09-01

    The influence of two neonicotinoids, i.e., imidacloprid (IMI) and acetamiprid (ACE), on soil microbial activities was investigated in a short period of time using a combination of the microcalorimetric approach and enzyme tests. Thermodynamic parameters such as Q T (J g(-1) soil), ∆H met (kJ mol(-1)), J Q/S (J g(-1) h(-1)), k (h(-1)), and soil enzymatic activities, dehydrogenase, phosphomonoesterase, arginine deaminase, and urease, were used to evaluate whole metabolic activity changes and acute toxicity following IMI and ACE treatment. Various profiles of thermogenic curves reflect different soil microbial activities. The microbial growth rate constant k, total heat evolution Q T (expect for IMI), and inhibitory ratio I show linear relationship with the doses of IMI and ACE. Q T for IMI increases at 0.0-20 μg g(-1) and then decreases at 20-80 μg g(-1), possibly attributing to the presence of tolerant microorganisms. The 50 % inhibitory ratios (IC50) of IMI and ACE are 95.7 and 77.2 μg g(-1), respectively. ACE displays slightly higher toxicity than IMI. Plots of k and Q T against microbial biomass-C indicate that the k and Q T are growth yield-dependent. IMI and ACE show 29.6; 40.4 and 23.0; and 23.3, 21.7, and 30.5 % inhibition of dehydrogenase, phosphomonoesterase, and urease activity, respectively. By contrast, the arginine deaminase activity is enhanced by 15.2 and 13.2 % with IMI and ACE, respectively. The parametric indices selected give a quantitative dose-response relationship of both insecticides and indicate that ACE is more toxic than IMI due to their difference in molecular structures.

  20. Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro.

    PubMed

    Bai, Yajuan; Liu, Lei; Zhang, Ruifen; Huang, Fei; Deng, Yuanyuan; Zhang, Mingwei

    2017-03-01

    An extraction method employing ultrahigh pressure-assisted enzymatic treatment was developed and optimized by response surface methodology to increase the yield of longan pulp polysaccharides (LP-UE). A maximum polysaccharides yield of 8.55% was obtained under the optimal conditions of 407MPa ultrahigh pressure maintained for 6min with an enzyme to pretreated material ratio of 1:100, an enzymolysis time of 1.7h and a water to pretreated material ratio of 42ml/g. Subsequently, the physicochemical properties and acetylcholinesterase (AChE) inhibitory activity of LP-UE were compared to those of longan pulp polysaccharides (LP) extracted by hot water (LP-H), ultrahigh pressure (LP-U) or enzymatic treatment (LP-E). Results demonstrated that the extraction yield, hexuronic acid content and AChE inhibitory activity of LP-UE was the highest among the four LP samples. LP-UE was primarily made up of arabinose, glucose, and galactose and was linked mainly by β-type glycosidic linkage. The FTIR spectrum of LP-UE was very similar to those of LP-H, LP-U, and LP-E. In summary, ultrahigh pressure-assisted enzymatic treatment is a more efficient technique for extracting LP with considerable improvement of both yield and memory enhancement function.

  1. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  2. Priming effects and enzymatic activity in Israeli soils under treated wastewater and freshwater irrigation

    NASA Astrophysics Data System (ADS)

    Anissimova, Marina; Heinze, Stefanie; Chen, Yona; Tarchitzky, Jorge; Marschner, Bernd

    2014-05-01

    Irrigation of soils with treated wastewater (TWW) directly influences microbial processes of soil. TWW contains easily decomposable organic material, which can stimulate the activity of soil microorganisms and, as a result, lead to the excessive consumption of soil organic carbon pool. We investigated the effects of irrigation with TWW relative to those of irrigation with freshwater (FW) on the microbial parameters in soils with low (7%) and medium (13%) clay content in a lysimeter experiment. The objectives of our study were to (i) determine the impact of water quality on soil respiration and enzymatic activity influenced by clay content and depth, and (ii) work out the changes in the turnover of soil organic matter (PE, priming effects). Samples were taken from three soil depths (0-10, 10-20, and 40-60 cm). Soil respiration and PE were determined in a 21-days incubation experiment after addition of uniformly 14C-labeled fructose. Activity of 10 extracellular enzymes (EEA, from C-, N-, P-, and S-cycle), phenol oxidase and peroxidase activity (PO+PE), and dehydrogenase activity (DHA) were assayed. Microbial Community-Level Physiological Profiles (CLPP) using four substrates, and microbial biomass were determined. The results showed that the clay content acted as the main determinative factor. In the soil with low clay content the water quality had a greater impact: the highest PE (56%) was observed in the upper layer (0-10cm) under FW irrigation; EEA of C-, P-, and S-cycles was significantly higher in the upper soil layer under TWW irrigation. Microbial biomass was higher in the soil under TWW irrigation and decreased with increasing of depth (50 μg/g soil in the upper layer, 15 μg/g soil in the lowest layer). This tendency was also observed for DHA. Contrary to the low clay content, in the soil with medium clay content both irrigation types caused the highest PE in the lowest layer (65% under FW irrigation, 48% under TWW irrigation); the higher substrate

  3. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  4. fMRI activation patterns in an analytic reasoning task: consistency with EEG source localization

    NASA Astrophysics Data System (ADS)

    Li, Bian; Vasanta, Kalyana C.; O'Boyle, Michael; Baker, Mary C.; Nutter, Brian; Mitra, Sunanda

    2010-03-01

    Functional magnetic resonance imaging (fMRI) is used to model brain activation patterns associated with various perceptual and cognitive processes as reflected by the hemodynamic (BOLD) response. While many sensory and motor tasks are associated with relatively simple activation patterns in localized regions, higher-order cognitive tasks may produce activity in many different brain areas involving complex neural circuitry. We applied a recently proposed probabilistic independent component analysis technique (PICA) to determine the true dimensionality of the fMRI data and used EEG localization to identify the common activated patterns (mapped as Brodmann areas) associated with a complex cognitive task like analytic reasoning. Our preliminary study suggests that a hybrid GLM/PICA analysis may reveal additional regions of activation (beyond simple GLM) that are consistent with electroencephalography (EEG) source localization patterns.

  5. Consistency in boldness, activity and exploration at different stages of life

    PubMed Central

    2013-01-01

    Background Animals show consistent individual behavioural patterns over time and over situations. This phenomenon has been referred to as animal personality or behavioural syndromes. Little is known about consistency of animal personalities over entire life times. We investigated the repeatability of behaviour in common voles (Microtus arvalis) at different life stages, with different time intervals, and in different situations. Animals were tested using four behavioural tests in three experimental groups: 1. before and after maturation over three months, 2. twice as adults during one week, and 3. twice as adult animals over three months, which resembles a substantial part of their entire adult life span of several months. Results Different behaviours were correlated within and between tests and a cluster analysis showed three possible behavioural syndrome-axes, which we name boldness, exploration and activity. Activity and exploration behaviour in all tests was highly repeatable in adult animals tested over one week. In animals tested over maturation, exploration behaviour was consistent whereas activity was not. Voles that were tested as adults with a three-month interval showed the opposite pattern with stable activity but unstable exploration behaviour. Conclusions The consistency in behaviour over time suggests that common voles do express stable personality over short time. Over longer periods however, behaviour is more flexible and depending on life stage (i.e. tested before/after maturation or as adults) of the tested individual. Level of boldness or activity does not differ between tested groups and maintenance of variation in behavioural traits can therefore not be explained by expected future assets as reported in other studies. PMID:24314274

  6. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    PubMed

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems.

  7. A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans

    PubMed Central

    Butler, Victoria J.; Branicky, Robyn; Yemini, Eviatar; Liewald, Jana F.; Gottschalk, Alexander; Kerr, Rex A.; Chklovskii, Dmitri B.; Schafer, William R.

    2015-01-01

    Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force–posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control. PMID:25551155

  8. A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

    PubMed

    Butler, Victoria J; Branicky, Robyn; Yemini, Eviatar; Liewald, Jana F; Gottschalk, Alexander; Kerr, Rex A; Chklovskii, Dmitri B; Schafer, William R

    2015-01-06

    Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

  9. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity

    PubMed Central

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui

    2017-01-01

    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL. PMID:28194412

  10. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity.

    PubMed

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui; Meng, Zhaohui

    2017-01-01

    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL.

  11. Second-Order Perturbation Theory for Generalized Active Space Self-Consistent-Field Wave Functions.

    PubMed

    Ma, Dongxia; Li Manni, Giovanni; Olsen, Jeppe; Gagliardi, Laura

    2016-07-12

    A multireference second-order perturbation theory approach based on the generalized active space self-consistent-field (GASSCF) wave function is presented. Compared with the complete active space (CAS) and restricted active space (RAS) wave functions, GAS wave functions are more flexible and can employ larger active spaces and/or different truncations of the configuration interaction expansion. With GASSCF, one can explore chemical systems that are not affordable with either CASSCF or RASSCF. Perturbation theory to second order on top of GAS wave functions (GASPT2) has been implemented to recover the remaining electron correlation. The method has been benchmarked by computing the chromium dimer ground-state potential energy curve. These calculations show that GASPT2 gives results similar to CASPT2 even with a configuration interaction expansion much smaller than the corresponding CAS expansion.

  12. Long-term effects of fertilizer on soil enzymatic activity of wheat field soil in Loess Plateau, China.

    PubMed

    Hu, Weigang; Jiao, Zhifang; Wu, Fasi; Liu, Yongjun; Dong, Maoxing; Ma, Xiaojun; Fan, Tinglu; An, Lizhe; Feng, Huyuan

    2014-12-01

    The effects of long-term (29 years) fertilization on local agro-ecosystems in the Loess Plateau of northwest China, containing a single or combinations of inorganic (Nitrogen, N; Phosphate, P) and organic (Mature, M Straw, S) fertilizer, including N, NP, SNP, M, MNP, and a control. The soil enzymes, including dehydrogenase, urease, alkaline phosphatase, invertase and glomalin, were investigated in three physiological stages (Jointing, Dough, and Maturity) of wheat growth at three depths of the soil profile (0-15, 16-30, 31-45 cm). We found that the application of farmyard manure and straw produced the highest values of soil enzymatic activity, especially a balanced applied treatment of MNP. Enzymatic activity was lowest in the control. Values were generally highest at dough, followed by the jointing and maturity stages, and declined with soil profile depth. The activities of the enzymes investigated here are significantly correlated with each other and are correlated with soil nutrients, in particular with soil organic carbon. Our results suggest that a balanced application of fertilizer nutrients and organic manure (especially those containing P) has positive effects on multiple soil chemical parameters, which in turn enhances enzyme activity. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, as its application can result in a substantial increase in agricultural production and can be sustainable for many years.

  13. Enzymatic Activity of the Soybean Ecto-Apyrase GS52 Is Essential for Stimulation of Nodulation1[W][OA

    PubMed Central

    Tanaka, Kiwamu; Nguyen, Cuong T.; Libault, Marc; Cheng, Jianlin; Stacey, Gary

    2011-01-01

    Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation. PMID:21346172

  14. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  15. Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

    PubMed Central

    Orencio-Trejo, Montserrat; Flores, Noemí; Escalante, Adelfo; Hernández-Chávez, Georgina; Bolívar, Francisco; Gosset, Guillermo; Martinez, Alfredo

    2008-01-01

    Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates. PMID:18471274

  16. A priori complete active space self consistent field localized orbitals: an application on linear polyenes

    NASA Astrophysics Data System (ADS)

    Angeli, Celestino; Sparta, Manuel; Cimiraglia, Renzo

    2006-03-01

    A recently proposed a priori localization technique is used to exploit the possibility to reduce the number of active orbitals in a Complete Active Space Self Consistent Field calculation. The work relies on the fact that the new approach allows a strict control on the nature of the active orbitals and therefore makes it possible to include in the active space only the relevant orbitals. The idea is tested on the calculation of the energy barrier for rigid rotation of linear polyenes. In order to obtain a relevant set of data, a number of possible rotations around double bonds have been considered in the ethylene, butadiene, hexatriene, octatetraene, decapentaene, dodecahexaene molecules. The possibility to reduce the dimension of the active space has been investigated, considering for each possible rotation different active spaces ranging from the minimal dimension of 2 electrons in 2 π orbitals to the π-complete space. The results show that the rigid isomerization in the polyene molecules can be described with a negligible loss in accuracy with active spaces no larger than ten orbitals and ten electrons. In the special case of the rotation around the terminal double bond, the space can be further reduced to six orbitals and six electrons with a large decrease of the computational cost. An interesting summation rule has been found and verified for the stabilization of the energy barriers as a function of the dimension of the conjugated lateral chains and of the dimension of the active space.

  17. Discovery of Cell-Type-Specific and Disease-Related Enzymatic Activity Changes via Global Evaluation of Peptide Metabolism.

    PubMed

    Onagi, Jun; Komatsu, Toru; Ichihashi, Yuki; Kuriki, Yugo; Kamiya, Mako; Terai, Takuya; Ueno, Tasuku; Hanaoka, Kenjiro; Matsuzaki, Hiroyuki; Hata, Keisuke; Watanabe, Toshiaki; Nagano, Tetsuo; Urano, Yasuteru

    2017-03-08

    Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.

  18. Quantitative Collection and Enzymatic Activity of Glucose Oxidase Nanotubes Fabricated by Templated Layer-by-Layer Assembly.

    PubMed

    Zhang, Shouwei; Demoustier-Champagne, Sophie; Jonas, Alain M

    2015-08-10

    We report on the fabrication of enzyme nanotubes in nanoporous polycarbonate membranes via the layer-by-layer (LbL) alternate assembly of polyethylenimine (PEI) and glucose oxidase (GOX), followed by dissolution of the sacrificial template in CH2Cl2, collection, and final dispersion in water. An adjuvant-assisted filtration methodology is exploited to extract quantitatively the nanotubes without loss of activity and morphology. Different water-soluble CH2Cl2-insoluble adjuvants are tested for maximal enzyme activity and nanotube stability; whereas NaCl disrupts the tubes by screening electrostatic interactions, the high osmotic pressure created by fructose also contributes to loosening the nanotubular structures. These issues are solved when using neutral, high molar mass dextran. The enzymatic activity of intact free nanotubes in water is then quantitatively compared to membrane-embedded nanotubes, showing that the liberated nanotubes have a higher catalytic activity in proportion to their larger exposed surface. Our study thus discloses a robust and general methodology for the fabrication and quantitative collection of enzymatic nanotubes and shows that LbL assembly provides access to efficient enzyme carriers for use as catalytic swarming agents.

  19. Modulation of enzymatic activity of Src-family kinases in bovine T cells transformed by Theileria parva.

    PubMed

    Fich, C; Klauenberg, U; Fleischer, B; Bröker, B M

    1998-08-01

    After infection with sporozoites of the protozoon Theileria parva (Tp) bovine T cells are readily transformed to permanent growth in vivo and in vitro. Their transformed state depends on the constant presence of the parasite but membrane signals remain important. Non-receptor tyrosine kinases play a critical role in the transduction of membrane signals in haematopoietic cells. We have investigated Src-family kinases in bovine T cells transformed by Tp. The T cell receptor-associated tyrosine kinase p60fyn had high activity in all cell lines tested. In addition, weak phosphorylation of 2 novel bands was observed associated with Fyn. In contrast to Fyn, enzymatic activity of p56lck, which in T cells has an essential role in signalling, was low. Furthermore, 1 of 3 Tp transformed cell lines was completely devoid of p56lck indicating that the enzyme is not necessary for the Tp dependent growth of the T cells. In addition to p60fyn and p56lck weak enzymatic activity of 1 splice variant of p53/56lyn was observed after infection of T cells with Tp. These data show that growth transformation by Tp influences kinase activity in bovine T cells. However, they also prove that p56lck does not play an essential role in the transformation mechanism.

  20. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  1. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

    PubMed

    Andjelković, Ana; Oliveira, Marcos T; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T

    2015-12-17

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

  2. Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

    NASA Astrophysics Data System (ADS)

    Wang, Gang; Yau, Siu-Tung

    2005-12-01

    The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

  3. Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity*

    PubMed Central

    Hughes, Siobhan; Jenkins, Victoria; Dar, Mohd Jamal; Engelman, Alan; Cherepanov, Peter

    2010-01-01

    Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK. PMID:19864417

  4. Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis.

    PubMed

    van der Geld, Y M; Tool, A T J; Videler, J; de Haas, M; Tervaert, J W Cohen; Stegeman, C A; Limburg, P C; Kallenberg, C G M; Roos, D

    2002-09-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.

  5. Effect of high compost temperature on enzymatic activity and species diversity of culturable bacteria in cattle manure compost.

    PubMed

    Miyatake, Fumihito; Iwabuchi, Kazunori

    2005-11-01

    To clarify the characteristics of thermophilic bacteria in cattle manure compost, enzymatic activity and species diversity of cultivated bacteria were investigated at 54, 60, 63, 66 and 70 degrees C, which were dependent on composting temperature. The highest level of thermophilic bacterial activity was observed at 54 degrees C. Following an increase in temperature to 63 degrees C, a reduction in bacterial diversity was observed. At 66 degrees C, bacterial diversity increased again, and diverse bacteria including Thermus spp. and thermophilic Bacillus spp. appeared to adapt to the higher temperature. At 70 degrees C, bacterial activity measured as superoxide dismutase and catalase activity was significantly higher than at 66 degrees C. However, the decomposition rate of protein in the compost was lower than the rate at 66 degrees C due to the higher compost temperature.

  6. Enhancement of antibiotic-activity through complexation with metal ions - Combined ITC, NMR, enzymatic and biological studies.

    PubMed

    Möhler, Jasper S; Kolmar, Theresa; Synnatschke, Kevin; Hergert, Marcel; Wilson, Liam A; Ramu, Soumya; Elliott, Alysha G; Blaskovich, Mark A T; Sidjabat, Hanna E; Paterson, David L; Schenk, Gerhard; Cooper, Matthew A; Ziora, Zyta M

    2017-02-01

    Alternative solutions need to be developed to overcome the growing problem of multi-drug resistant bacteria. This study explored the possibility of creating complexes of antibiotics with metal ions, thereby increasing their activity. Analytical techniques such as isothermal titration calorimetry and nuclear magnetic resonance were used to examine the structure and interactions between Cu(II), Ag(I) or Zn(II) and β-lactam antibiotics. The metal-β-lactam complexes were also tested for antimicrobial activity, by micro-broth dilution and disk diffusion methods, showing a synergistic increase in the activity of the drugs, and enzymatic inhibition assays confirming inhibition of β-lactamases responsible for resistance. The metal-antibiotic complex concept was proven to be successful with the activity of the drugs enhanced against β-lactamase-producing bacteria. The highest synergistic effects were observed for complexes formed with Ag(I).

  7. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    NASA Astrophysics Data System (ADS)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P < 0.05). In the hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P < 0.05). Glutathione peroxidase activity did not change. Relative gene expression of catalase and superoxide dismutases (1 and 2) was downregulated, while glutathione peroxidase was upregulated in the brain of the cold-induced pulmonary hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  8. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils

    PubMed Central

    2012-01-01

    Background Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation. Methods In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran) and their growth ability was checked in potato dextrose agar (PDA) media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase) was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w). Results Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected as the most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed the highest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp., Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively. Conclusions Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities. PMID:23369665

  9. Enzymatically-Processed Wheat Bran Enhances Macrophage Activity and Has in Vivo Anti-Inflammatory Effects in Mice

    PubMed Central

    Kang, Hee; Lee, Mi-Gi; Lee, Jae-Kang; Choi, Yong-Hyun; Choi, Yong-Seok

    2016-01-01

    Wheat bran is a rich source of dietary fiber, of which arabinoxylan is the most abundant non-starch polysaccharide. Arabinoxylan has been known to exert in vivo immunological activities. Based on prior findings, we pretreated wheat bran with enzymatic hydrolysis to increase the release of soluble arabinoxylan and investigated whether oral administration of wheat bran altered macrophage activity in a mouse model. After four weeks of treatment, we isolated peritoneal macrophages for phagocytic receptor analysis and lipopolysaccharide (LPS)-induced inflammatory changes. In the second experiment, mice given wheat bran were intraperitoneally stimulated with LPS and serum levels of pro- and anti-inflammatory cytokines were determined. The expression of SRA and CD36, and phagocytic activity increased (p < 0.05, respectively). Ex vivo stimulation of macrophages by LPS resulted in reduced surface expression of CD40 (p < 0.05) and decreased production of nitric oxide (p < 0.005), tumor necrosis factor (TNF)-α (p < 0.005), interleukin (IL)-6 (p < 0.01), and IL-12 (p < 0.05). Mice treated with wheat bran showed decreased levels of serum TNF-α and IL-6 (p < 0.05, respectively) and an increased level of serum anti-inflammatory IL-10 (p < 0.05) in response to intraperitoneal LPS. Enzymatically-processed wheat bran boosts macrophage phagocytic capacity possibly through up-regulation of scavenger receptors and confers anti-inflammatory effects, indicating its potential as an immuno-enhancing functional food. PMID:27043618

  10. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity

    PubMed Central

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-01-01

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution. PMID:28186169

  11. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity.

    PubMed

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-02-10

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution.

  12. Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol.

    PubMed

    Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar

    2016-02-01

    Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment.

  13. Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid.

    PubMed

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-15

    Ferredoxin:NADP(+) oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP(+)), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

  14. In Vitro Enzymatic Activities of Bacteriochlorophyll a Synthase Derived from the Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum.

    PubMed

    Saga, Yoshitaka; Hirota, Keiya; Harada, Jiro; Tamiaki, Hitoshi

    2015-08-18

    The activity of an enzyme encoded by the CT1610 gene in the green sulfur photosynthetic bacterium Chlorobaculum tepidum, which was annotated as bacteriochlorophyll (BChl) a synthase, BchG (denoted as tepBchG), was examined in vitro using the lysates of Escherichia coli containing the heterologously expressed enzyme. BChl a possessing a geranylgeranyl group at the 17-propionate residue (BChl aGG) was produced from bacteriochlorophyllide (BChlide) a and geranylgeranyl pyrophosphate in the presence of tepBchG. Surprisingly, tepBchG catalyzed the formation of BChl a bearing a farnesyl group (BChl aF) as in the enzymatic production of BChl aGG, indicating loose recognition of isoprenoid pyrophosphates in tepBchG. In contrast to such loose recognition of isoprenoid substrates, BChlide c and chlorophyllide a gave no esterifying product upon being incubated with geranylgeranyl or farnesyl pyrophosphate in the presence of tepBchG. These results confirm that tepBchG undoubtedly acts as the BChl a synthase in Cba. tepidum. The enzymatic activity of tepBchG was higher than that of BchG of Rhodobacter sphaeroides at 45 °C, although the former activity was lower than the latter below 35 °C.

  15. Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

    NASA Astrophysics Data System (ADS)

    Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

    2001-12-01

    Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

  16. Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.

    PubMed

    Fu, Xin-Miao; Zhu, Bao Ting

    2010-07-01

    Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

  17. Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase.

    PubMed

    Shelat, Nirav Y; Parhi, Sidhartha; Ostermeier, Marc

    2017-02-01

    Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1α. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.

  18. Effects of cerium oxide nanoparticles on soil enzymatic activities and wheat grass nutrients uptake

    NASA Astrophysics Data System (ADS)

    Li, Biting; Chen, Yirui; Bai, Lingyun; Jacobson, Astrid; Darnault, Christophe

    2015-04-01

    The US National Science Foundation estimated that the use of nanomaterials and nanotechnology would reach a global market value of 1 million this year. Concomitant with the wide applications of nanoparticles is an increasing risk of adverse effects to the environment and human health. As a common nanomaterial used as a fuel catalyst and polish material, cerium (IV) oxide nanoparticles (CeO2 NP) were tested for their potential impact on soil health and plant growth. Through exposure by air, water, and solid deposition, nanoparticles may accumulate in soils and impact agricultural systems. The objectives of this research were to determine whether CeO2 NPs affect the growth of wheat grass and selected soil enzyme activities chose as indicators of soil health. Wheat grass was grown in plant boxes containing CeO2 NPs mixed with agricultural soil at different concentrations. Two control groups were included: one consisting of soil with plants but no CeO2 NPs, and one containing only soil, i.e., no NP or wheat plants added. The plants were grown for 10 weeks and harvested every two weeks in a laboratory under sodium growth lights. At the end of the each growing period, two weeks, soils were assayed for phosphatase, β-glucosidase, and urease activities, and NPK values. Spectrophotometer analyses were used to assess enzyme activities, and NPK values were tested by Clemson Agricultural Center. Wheat yields were estimated by shoot and root lengths and weights.

  19. Antiproliferative and GH-inhibitory activity of chimeric peptides consisting of GHRP-6 and somatostatin.

    PubMed

    Dasgupta, P; Singh, A T; Mukherjee, R

    1999-06-07

    Chimeric peptides consisting of growth hormone releasing peptide (GHRP-6) linked to somatostatin (6-11) via an amide bond to provide the effector parts of both the peptides were synthesized. The anti-proliferative, cytotoxic, and GH-inhibitory activities of these chimeric peptides were determined in vitro in the rat pituitary adenoma cell line GH3. One of the chimeric peptides, GSD, exhibited significantly greater (p < 0.001) anti-neoplastic and GH-inhibitory activity, as compared to RC-160. The hybrid peptides displayed high affinity binding to somatostatin receptors on GH3 cells. The bioactivity of GSD was found to be mediated by the stimulation of tyrosine phosphatase, involving a cGMP-dependent pathway, through pertussis toxin-sensitive G-proteins. Such potent GH-inhibitory chimeric peptides may be of potential importance in the therapy of acromegaly, as well as provide novel tools to study the regulation of GH secretion by GHRP and somatostatin.

  20. Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis.

    PubMed

    Aoyama, Masato; Kawada, Tsuyoshi; Satake, Honoo

    2012-03-01

    We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.

  1. Amifostine Induces Antioxidant Enzymatic Activities in Normal Tissues and a Transplantable Tumor That Can Affect Radiation Response

    SciTech Connect

    Grdina, David J. Murley, Jeffrey S.; Kataoka, Yasushi; Baker, Kenneth L.; Kunnavakkam, Rangesh; Coleman, Mitchell C.; Spitz, Douglas R.

    2009-03-01

    Purpose: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect. Methods and Materials: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined. Results: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes. Conclusions: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

  2. Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

    PubMed

    Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

    2013-12-01

    This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like.

  3. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    SciTech Connect

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  4. Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner.

    PubMed

    Lee, Phil Young; Kho, Chang Won; Lee, Do Hee; Kang, Sunghyun; Kang, Seongman; Lee, Sang Chul; Park, Byoung Chul; Cho, Sayeon; Bae, Kwang-Hee; Park, Sung Goo

    2007-10-19

    Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.

  5. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils.

    PubMed

    Sahoo, Ranjan Kumar; Tuteja, Narendra

    2013-08-01

    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community.

  6. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  7. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils

    PubMed Central

    Sahoo, Ranjan Kumar; Tuteja, Narendra

    2013-01-01

    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community. PMID:23733066

  8. The impact of element-element interactions on antioxidant enzymatic activity in the blood of white stork (Ciconia ciconia) chicks.

    PubMed

    Kamiński, Piotr; Kurhalyuk, Nataliya; Kasprzak, Mariusz; Jerzak, Leszek; Tkachenko, Halyna; Szady-Grad, Małgorzata; Klawe, Jacek J; Koim, Beata

    2009-02-01

    The aim of this work was to determine interrelationships among macroelements Na, K, Ca, Mg, and Fe, microelements Zn, Cu, Mn, and Co, and toxic heavy metals Pb and Cd in the blood of white stork Ciconia ciconia, during postnatal development, in different Polish environments, and their impact on the activity of antioxidant enzymes. We considered the content of thiobarbituric acid-reactive substances (TBARSs), i.e., malondialdehyde (MDA), and activity of superoxide dismutase (SOD), catalase (CAT), ceruloplasmine (CP), glutathione peroxidase (GPx), and glutathione reductase (GR). Blood samples were collected from storks developing at Odra meadows (Kłopot; southwestern Poland). They were compared with blood of chicks from several suburban sites located 20 km away from Zielona Góra (0.1 million inhabitants; southwestern Poland) and near Głogów, where a copper smelter is situated. We also conducted research in the Pomeranian region (Cecenowo; northern Poland). We collected blood samples via venipuncture of the brachial vein of chicks in 2005-2007. They were retrieved from the nest and placed in individual ventilated cotton sacks. The blood was collected using a 5-ml syringe washed with ethylenediaminetetraacetic acid (EDTA). We found significant interactions between macro- and microelements and enzymatic activity and TBARS products. We noticed the predominance of Cd and Pb participation in element-enzyme interactions. Simultaneously, we found interrelationships between cadmium and Na, K, Ca, Mg, and Fe and the activity of antioxidant enzymes SOD, CAT, CP, GR, and TBARS products in the blood of white stork chicks. In the case of lead these relationships were not numerous and they were significant for Ca, Mg, Cu, Mn, and Co. Correlations with enzymes were significant for Pb-CAT and Pb-TBARS. We noted that activities of most enzymes (SOD, CAT, CP, GR) and TBARS products are determined by their interactions with physiological elements Na, Ca, Mg, Fe, and Zn and toxic

  9. Effects of 24-epibrassinolide on enzymatic browning and antioxidant activity of fresh-cut lotus root slices.

    PubMed

    Gao, Hui; Chai, HongKang; Cheng, Ni; Cao, Wei

    2017-02-15

    Fresh-cut lotus root slices were treated with 80nM 24-epibrassinolide (EBR) and then stored at 4°C for 8days to investigate the effects on cut surface browning. The results showed that EBR treatment reduced cut surface browning in lotus root slices and alleviated membrane lipid peroxidation as reflected by low malondialdehyde content and lipoxygenase activity. EBR treatment inhibited the activity of phenylalanine ammonia lyase and polyphenol oxidase, and subsequently decreased phenolics accumulation and soluble quniones formation. The treatment also stimulated the activity of peroxidase, catalase and ascorbate peroxidase and delayed the loss of ascorbic acid, which would help prevent membrane lipid peroxidation, as a consequence, reducing decompartmentation of enzymes and substrates causing enzymatic browning. These results indicate that EBR treatment is a promising attempt to control browning at cut surface of fresh-cut lotus root slices.

  10. Bacterial Production and Enzymatic Activities in Deep-Sea Sediments of the Pacific Ocean: Biogeochemical Implications of Different Temperature Constraints

    NASA Astrophysics Data System (ADS)

    Danovaro, R.; Corinaldesi, C.; dell'Anno, A.

    2002-12-01

    The deep-sea bed, acting as the ultimate sink for organic material derived from the upper oceans primary production, is now assumed to play a key role in biogeochemical cycling of organic matter on global scale. Early diagenesis of organic matter in marine sediments is dependent upon biological processes (largely mediated by bacterial activity) and by molecular diffusion. Organic matter reaching the sea floor by sedimentation is subjected to complex biogeochemical transformations that make organic matter largely unsuitable for direct utilization by benthic heterotrophs. Extracellular enzymatic activities in the sediment is generally recognized as the key step in the degradation and utilization of organic polymers by bacteria and a key role in biopolymeric carbon mobilization is played by aminopeptidase, alkaline phosphatase and glucosidase activities. In the present study we investigated bacterial density, bacterial C production and exo-enzymatic activities (aminopeptidase, glucosidase and phosphatase activity) in deep-sea sediments of the Pacific Ocean in relation with the biochemical composition of sediment organic matter (proteins, carbohydrates and lipids), in order to gather information on organic matter cycling and diagenesis. Benthic viral abundance was also measured to investigate the potential role of viruses on microbial loop functioning. Sediment samples were collected at eight stations (depth ranging from 2070-3100 m) along two transects located at the opposite side (north and south) of ocean seismic ridge Juan Fernandez (along latitudes 33° 20' - 33° 40'), constituted by the submerged vulcanoes, which connects the Chilean coasts to Rapa Nui Island. Since the northern and southern sides of this ridge apparently displayed small but significant differences in deep-sea temperature (related to the general ocean circulation), this sampling strategy allowed also investigating the role of different temperature constraints on bacterial activity and

  11. A Self-Consistent Radiative Transfer Model for Simulating Active and Passive Observations of Precipitation

    NASA Astrophysics Data System (ADS)

    Adams, I. S.

    2015-12-01

    Current generation sensors suites such as those included on the Global Precipitation Measurement (GPM) mission, Aquarius, and Soil Moisture Active / Passive (SMAP) exploit a combination to provide a greater understanding of geophysical phenomena. While "operationalized" retrieval algorithms require fast forward models, the ability to perform higher fidelity simulations is necessary for understanding the physics of remote sensing problems to test assumptions and to develop parameterizations for the fast models. To ensure proper synergy between active and passive modeling, forward models must be consistent between the two sensor types. This work presents a self-consistent active and passive radiative transfer model for simulating radar and radiometer responses to precipitation. To accomplish this, we extend the Atmospheric Radiative Transfer Simulator (ARTS) version 2.3 to solve the radiative transfer equation for radar under multiple scattering conditions using Monte Carlo integration. Early versions of ARTS (1.1 and later) included a passive Monte Carlo solver, and ARTS is capable of handling atmospheres of up to three dimensions with ellipsoidal planetary geometries. The modular nature of ARTS facilitates extensibility, and the well-developed ray-tracing tools are suited for implementation of Monte Carlo algorithms. Finally, since ARTS handles the full Stokes vector, co- and cross-polarized reflectivity products are possible for scenarios that include nonspherical particles, with or without preferential alignment. The accuracy of the forward model will be demonstrated, and the effects of multiple scattering will be detailed. The three-dimensional nature of the radiative transfer model will be useful for understanding the effects of nonuniform beamfill and multiple scattering for spatially heterogeneous precipitation events. This targets of this forward model are GPM (the Dual-wavelength Precipitation Radar (DPR) and GPM Microwave Imager (GMI)) and airborne sensors

  12. [Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)].

    PubMed

    Chaparro, Deisy Fernanda; Rosas, Diana Carolina; Varela, Amanda

    2009-12-31

    White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.

  13. Alternations in the liver enzymatic activity of Common carp, Cyprinus carpio in response to parasites, Dactylogyrus spp. and Gyrodactylus spp.

    PubMed

    Rastiannasab, Abulhasan; Afsharmanesh, Shiva; Rahimi, Ruhollah; Sharifian, Iman

    2016-12-01

    The present study was carried out to investigate the effects of parasites, monogenea, Dactylogyrus spp. and Gyrodactylus spp. on some enzymatic and biochemical components of liver in healthy and infected common carp, Cyprinus carpio. For this purpose, 10 healthy and 10 infected fish were collected from farm. The blood samples were taken and after separation of serum, the values of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) enzymes activities as well as Creatinine and Urea were measured. Based on obtained results, the values of AST, ALT enzymes activities as well as Creatinine and Urea were higher in the infected fish compared to non-infected fish. In conclusion; our results reveals that infection with external parasites, Dactylogyrus spp. and Gyrodactylus spp. can causes some dysfunctions in liver and kidney of common carp.

  14. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

  15. Self-consistent simulation of CdTe solar cells with active defects

    DOE PAGES

    Brinkman, Daniel; Guo, Da; Akis, Richard; ...

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Lastly, we will give numerical results comparing our results to known 1D simulations tomore » demonstrate the accuracy of the solver and then show results unique to the 2D case.« less

  16. Self-consistent simulation of CdTe solar cells with active defects

    SciTech Connect

    Brinkman, Daniel; Guo, Da; Akis, Richard; Ringhofer, Christian; Sankin, Igor; Fang, Tian; Vasileska, Dragica

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Lastly, we will give numerical results comparing our results to known 1D simulations to demonstrate the accuracy of the solver and then show results unique to the 2D case.

  17. Self-consistent simulation of CdTe solar cells with active defects

    SciTech Connect

    Brinkman, Daniel; Ringhofer, Christian; Guo, Da; Akis, Richard; Vasileska, Dragica; Sankin, Igor; Fang, Tian

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Finally, we will give numerical results comparing our results to known 1D simulations to demonstrate the accuracy of the solver and then show results unique to the 2D case.

  18. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Tartrate Dehydratase from Bradyrhizobium japonicum

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Wood, B.; Almo, S.; Gerlt, J.

    2006-01-01

    We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second {beta}-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth -strands, respectively, as well as ligands for an essential Mg{sup 2+}, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth {beta}-strands, respectively. We screened a library of 46 acid sugars and discovered that only D-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (k{sub cat} = 7.3 s{sup -1}; k{sub cat}/K{sub M} = 8.5 x 10{sup 4} M{sup -1} s{sup -1}) are consistent with assignment of the D-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the {alpha}-proton to generate a Mg{sup 2+}-stabilized enediolate intermediate, and the vinylogous -elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by {sup 1}H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a 'simple' extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg{sup 2+}-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.

  19. Different enzymatic activities in carp (cyprinus carpio L.) as potential biomarkers of exposure to the pesticide methomyl.

    PubMed

    Hernández-Moreno, David; de la Casa-Resino, Irene; Maria Flores, José; González-Gómez, Manuel José; María Neila, Carlos; Soler, Francisco; Pérez-López, Marcos

    2014-09-29

    This study investigated the influence of the pesticide methomyl on different enzymatic activities in carp. The fish were exposed to a sub-lethal concentration (0.34 mg L-1) of methomyl for 15 days. On days 4 and 15, catalase (CAT) and glutathione-S-transferase (GST) activities were measured in the liver and gills. Acetylcholinesterase (AChE) activity in brain and muscle was also determined. Liver catalase activity slightly increased in exposed fish when compared to controls, but it was statistically significant only at the beginning of the experiment. No changes in CAT activity in the gills of exposed and control animals were observed (mean values were in the range 10.7-11.7 nmol min-1 per mg of protein). Liver GST activity was slightly inhibited in the exposed animals at the beginning of the study; however, it was significantly inhibited in the gills. Brain AChE activity was diminished throughout the experiment and significantly decreased after 96 h of exposure compared to controls (0.041 vs. 0.075 nmol min1 per mg of protein; p<0.001). Our findings suggest that CAT, GST, and AChE are reliable biomarkers of effect after exposure to methomyl.

  20. Amendment application in a multi-contaminated mine soil: effects on soil enzymatic activities and ecotoxicological characteristics.

    PubMed

    Manzano, Rebeca; Esteban, Elvira; Peñalosa, Jesús M; Alvarenga, Paula

    2014-03-01

    Several amendments were tested on soils obtained from an arsenopyrite mine, further planted with Arrhenatherum elatius and Festuca curvifolia, in order to assess their ability to improve soil's ecotoxicological characteristics. The properties used to assess the effects were: soil enzymatic activities (dehydrogenase, β-glucosidase, acid phosphatase, urease, protease and cellulase), terrestrial bioassays (Eisenia fetida mortality and avoidance behaviour), and aquatic bioassays using a soil leachate (Daphnia magna immobilisation and Vibrio fischeri bioluminescence inhibition). The treatment with FeSO4 1 % w/w was able to reduce extractable As in soil, but increased the extractable Cu, Mn and Zn concentrations, as a consequence of the decrease in soil pH, in relation to the unamended soil, from 5.0 to 3.4, respectively. As a consequence, this treatment had a detrimental effect in some of the soil enzymatic activities (e.g. dehydrogenase, acid phosphatase, urease and cellulase), did not allow plant growth, induced E. fetida mortality in the highest concentration tested (100 % w/w), and its soil leachate was very toxic towards D. magna and V. fischeri. The combined application of FeSO4 1 % w/w with other treatments (e.g. CaCO3 1 % w/w and paper mill 1 % w/w) allowed a decrease in extractable As and metals, and a soil pH value closer to neutrality. As a consequence, dehydrogenase activity, plant growth and some of the bioassays identified those as better soil treatments to this type of multi-contaminated soil.

  1. Lactones 42. Stereoselective enzymatic/microbial synthesis of optically active isomers of whisky lactone.

    PubMed

    Boratyński, Filip; Smuga, Małgorzata; Wawrzeńczyk, Czesław

    2013-11-01

    Two different methods, enzyme-mediated reactions and biotrasformations with microorganisms, were applied to obtain optically pure cis- and trans-isomers of whisky lactone 4a and 4b. In the first method, eight alcohol dehydrogenases were investigated as biocatalysts to enantioselective oxidation of racemic erythro- and threo-3-methyloctane-1,4-diols (1a and 1b). Oxidation processes with three of them, alcohol dehydrogenases isolated from horse liver (HLADH) as well as recombinant from Escherichia coli and primary alcohol dehydrogenase (PADH I), were characterized by the highest degree of conversion with moderate enantioselectivity (ee=27-82%) of the reaction. In all enzymatic reactions enantiomerically enriched not naturally occurring isomers of trans-(-)-(4R,5S)-4b or cis-(+)-(4R,5R)-4a were formed preferentially. In the second strategy, based on microbial lactonization of γ-oxoacids, naturally occurring opposite isomers of whisky lactones were obtained. Trans-(+)-(4S,5R)-isomer (ee=99%) of whisky lactone 4b was stereoselectively formed as the only product of biotransformations of 3-methyl-4-oxooctanoic acid (5) catalyzed by Didimospheria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp.1 KCH6670 and Saccharomyces cerevisiae AM464. Biotransformation of γ-oxoacid 5, in the culture of Beauveria bassiana AM278 and Pycnidiella resinae KCH50 afforded a mixtures of trans-(+)-(4S,5R)-4b with enantiomeric excess ee=99% and cis-(-)-(4S,5S)-4a with enantiomeric excesses ee=77% and ee=45% respectively.

  2. An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

    PubMed

    Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

    2017-02-01

    Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.

  3. Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

    PubMed

    Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

    2015-04-01

    Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

  4. Consistency tests in guaranteed simulation of nonlinear uncertain systems with application to an activated sludge process

    NASA Astrophysics Data System (ADS)

    Kletting, Marco; Rauh, Andreas; Aschemann, Harald; Hofer, Eberhard P.

    2007-02-01

    In this paper, interval arithmetic simulation techniques are presented to determine guaranteed enclosures of the state variables of both continuous and discrete-time systems with uncertain but bounded parameters. In nonlinear uncertain systems axis-parallel interval boxes are mapped to complexly shaped regions in the state space that represent sets of possible combinations of state variables. The approximation of each region by a single interval box causes an accumulating overestimation from time-step to time-step, usually called the wrapping effect. The algorithm presented in this paper minimizes the wrapping effect by applying consistency techniques based on interval Newton methods. Subintervals that do not belong to the exact solution at a given time can be eliminated in order to give a tighter but still conservative approximation of the exact solution. Additionally, efficient splitting and merging strategies are employed to limit the number of subintervals. The proposed algorithm is applied to the simulation of an activated sludge process in biological wastewater treatment.

  5. Enhancing phytochemical levels, enzymatic and antioxidant activity of spinach leaves by chitosan treatment and an insight into the metabolic pathway using DART-MS technique.

    PubMed

    Singh, Shachi

    2016-05-15

    Phytochemicals are health promoting compounds, synthesized by the plants to protect them against biotic or abiotic stress. The metabolic pathways leading to the synthesis of these phytochemicals are highly inducible; therefore methods could be developed to enhance their production by the exogenous application of chemical inducers/elicitors. In the present experiment, chitosan was used as an elicitor molecule to improve the phytochemical content of spinach plant. When applied at a concentration of 0.01 mg/ml as a foliar spray, chitosan was able to cause an increase in the enzymatic (peroxidase, catalase and phenylalanine ammonium lyase (PAL)) and non enzymatic (total phenolics, flavonoids and proteins) defensive metabolites, as well as, in the total antioxidant activity of the spinach leaves. A 1.7-fold increase in the total phenolics, a 2-fold increase in total flavonoid and a 1.6-fold increase in total protein were achieved with the treatment. A higher level of enzymatic activity was observed with a 4-fold increase in peroxidase and approximately 3-fold increases in catalase and phenylalanine ammonium lyase activity. Antioxidant activity showed a positive correlation between phenolic compounds and the enzymatic activity. Direct analysis in real time mass spectrometry (DART-MS) was applied to generate the metabolite profile of control and treated leaves. DART analysis revealed the activation of phenylpropanoid pathway by chitosan molecule, targeting the synthesis of diverse classes of flavonoids and their glycosides. Important metabolites of stress response were also visible in the DART spectra, including proline and free sugars.

  6. Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry.

    PubMed

    Schieltz, David M; McWilliams, Lisa G; Kuklenyik, Zsuzsanna; Prezioso, Samantha M; Carter, Andrew J; Williamson, Yulanda M; McGrath, Sara C; Morse, Stephen A; Barr, John R

    2015-03-01

    The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.

  7. Trapping RNase A on MCM41 pores: effects on structure stability, product inhibition and overall enzymatic activity.

    PubMed

    Matlahov, Irina; Geiger, Yasmin; Goobes, Gil

    2014-05-21

    Catalytic activity of enzymes can be drastically modified by immobilization on surfaces of different materials. It is particularly effective when the dimensions of the biomolecules and adsorption sites on the material surfaces are commensurate. This can be utilized to hinder the biological activity of degradation enzymes and switch off undesired biological processes. Ribonucleases are particularly attractive targets for complete sequestration being efficient at disintegrating viable RNA molecules. Here we show that efficient quenching of ribonuclease A activity can be achieved by immobilization on the surface of MCM41 porous silica. Electron microscopy, isothermal titration calorimetry, differential scanning calorimetry and adsorption isotherm measurements of ribonuclease A on the MCM41 surface are used to demonstrate that the enzyme adsorbs on the external surface of the porous silica through electrostatic interactions that overcome the unfavorable entropy change as the protein gets trapped on the surface, and that immobilization shifts up its denaturation temperature by 20-25 °C. Real-time kinetic measurements, using single injection titration calorimetry, demonstrate that enzymatic activity towards hydrolysis of cyclic nucleotides is lowered by nearly two orders of magnitude on MCM41 and that active inhibition by the formed product is much less effective on the surface than in solution.

  8. Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry

    PubMed Central

    Schieltz, David M.; McWilliams, Lisa G.; Kuklenyik, Zsuzsanna; Prezioso, Samantha M.; Carter, Andrew J.; Williamson, Yulanda M.; McGrath, Sara C.; Morse, Stephen A.; Barr, John R.

    2016-01-01

    The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity. PMID:25576235

  9. Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats.

    PubMed

    Cai, Bingna; Chen, Hua; Sun, Han; Sun, Huili; Wan, Peng; Chen, Deke; Pan, Jianyu

    2015-11-01

    The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOC-treated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOC-supplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P < .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women.

  10. Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

    PubMed

    de Menezes, Yamara A S; Félix-Silva, Juliana; da Silva-Júnior, Arnóbio A; Rebecchi, Ivanise M M; de Oliveira, Adeliana S; Uchoa, Adriana F; Fernandes-Pedrosa, Matheus de F

    2014-03-21

    Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  11. Identification of an Iron-Sulfur Cluster That Modulates the Enzymatic Activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase*

    PubMed Central

    Del Vecchio, Mariangela; Pogni, Rebecca; Baratto, Maria Camilla; Nobbs, Angela; Rappuoli, Rino; Pizza, Mariagrazia; Balducci, Enrico

    2009-01-01

    In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys67 and Cys128 into serine caused a drastic reduction in the E420/E280 ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes. PMID:19744927

  12. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  13. Allopregnanolone prevents memory impairment: effect on mRNA expression and enzymatic activity of hippocampal 3-α hydroxysteroid oxide-reductase.

    PubMed

    Escudero, Carla; Casas, Sebastián; Giuliani, Fernando; Bazzocchini, Vanesa; García, Sebastián; Yunes, Roberto; Cabrera, Ricardo

    2012-02-10

    In this work we investigated how the neurosteroid allopregnanolone can modulate learning and memory processes. For this purpose, we used ovariectomized (OVX) rats subcutaneously injected with oestradiol benzoate (E) alone or E and progesterone (P). Then, rats were injected in dorsal hippocampus with allopregnanolone or vehicle. Animals were tested in inhibitory avoidance task (IA task). After behavioural test hippocampal mRNA expression and enzymatic activity of 3α-HOR, the enzyme responsible of allopregnanolone synthesis, were analysed. In IA task OVX-EP rats spent less time on platform, compared to those OVX or OVX-E. Regression analyses revealed that there was a significant negative relationship between E-P infusion and performance in this task. Pre-training allopregnanolone administration to OVX-EP rats increased the time spent on the platform. Interestingly, when enzymatic activity of 3α-HOR was tested, OVX-EP rats showed a significant decrease in the enzymatic activity, compared with OVX and OVX-E rats. In addition, OVX-EP group showed a significant increase in the enzymatic activity after intrahippocampal infusion of allopregnanolone. On the other hand, when mRNA expression of 3α-HOR was analysed no differences were observed when the hippocampal allopregnanolone injection was done. These results suggest that E and P have amnesic effects on female rats, being reversed by allopregnanolone through its modulation on hippocampal 3α-HOR activity.

  14. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT... bodies, river basins, boundaries under the State's coastal nonpoint pollution control program, or...

  15. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT... bodies, river basins, boundaries under the State's coastal nonpoint pollution control program, or...

  16. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT... bodies, river basins, boundaries under the State's coastal nonpoint pollution control program, or...

  17. Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread.

    PubMed

    Black, Brenna A; Zannini, Emanuele; Curtis, Jonathan M; Gänzle, Michael G

    2013-03-01

    Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C(18:1) fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter(-1). Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter(-1). L. hammesii accumulated the monohydroxy C(18:1) fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter(-1) (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.

  18. Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans.

    PubMed

    Salmi, Marko; Jalkanen, Sirpa

    2006-09-01

    Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

  19. Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein on phospholipid Langmuir monolayers.

    PubMed

    Caseli, Luciano; Oliveira, Rafael G; Masui, Douglas C; Furriel, Rosa P M; Leone, Francisco A; Maggio, Bruno; Zaniquelli, M Elisabete D

    2005-04-26

    The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.

  20. Dynamics of microbiological parameters, enzymatic activities and worm biomass production during vermicomposting of effluent treatment plant sludge of bakery industry.

    PubMed

    Yadav, Anoop; Suthar, S; Garg, V K

    2015-10-01

    This paper reports the changes in microbial parameters and enzymatic activities during vermicomposting of effluent treatment plant sludge (ETPS) of bakery industry spiked with cow dung (CD) by Eisenia fetida. Six vermibins containing different ratios of ETPS and CD were maintained under controlled laboratory conditions for 15 weeks. Total bacterial and total fungal count increased upto 7th week and declined afterward in all the bins. Maximum bacterial and fungal count was 31.6 CFU × 10(6) g(-1) and 31 CFU × 10(4) g(-1) in 7th week. Maximum dehydrogenase activity was 1921 μg TPF g(-1) h(-1) in 9th week in 100 % CD containing vermibin, whereas maximum urease activity was 1208 μg NH4 (-)N g(-1) h(-1) in 3rd week in 100 % CD containing vermibin. The enzyme activity and microbial counts were lesser in ETPS containing vermibins than control (100 % CD). The growth and fecundity of the worms in different vermibins were also investigated. The results showed that initially biomass and fecundity of the worms increased but decreased at the later stages due to non-availability of the palatable feed. This showed that quality and palatability of food directly affect biological parameters of the system.

  1. Time-dependent restricted-active-space self-consistent-field theory with space partition

    NASA Astrophysics Data System (ADS)

    Miyagi, Haruhide; Madsen, Lars Bojer

    2017-02-01

    Aiming at efficient numerical analysis of time-dependent (TD) many-electron dynamics of atoms involving multielectron continua, the TD restricted-active-space self-consistent-field theory with space partition (TD-RASSCF-SP) is presented. The TD-RASSCF-SP wave function is expanded in terms of TD configuration-interaction coefficients with Slater determinants composed of two kinds of TD orbitals: M ̂ orbitals are defined to be nonvanishing in the inner region (V ̂), a small volume around the atomic nucleus, and M ˇ orbitals are nonvanishing in the large outer region (V ˇ). For detailed discussion of the SP strategy, the equations of motion are derived by two different formalisms for comparison. To ensure continuous differentiability of the wave function across the two regions, one of the formalisms makes use of the property of the finite-element discrete-variable-representation (FEDVR) functions and introduces additional time-independent orbitals. The other formalism is more general and is based on the Bloch operator as in the R -matrix theory, but turns out to be less practical for numerical applications. Hence, using the FEDVR-based formalism, the numerical performance is tested by computing double-ionization dynamics of atomic beryllium in intense light fields. To achieve high accuracy, M ̂ should be set large to take into account the strong many-electron correlation around the nucleus. On the other hand, M ˇ can be set much smaller than M ̂ for capturing the weaker correlation between the two outgoing photoelectrons. As a result, compared with more accurate multiconfigurational TD Hartree-Fock (MCTDHF) method, the TD-RASSCF-SP method may achieve comparable accuracy in the description of the double-ionization dynamics. There are, however, difficulties related to the stiffness of the equations of motion of the TD-RASSCF-SP method, which makes the required time step for this method smaller than the one needed for the MCTDHF approach.

  2. The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

    PubMed

    Xu; Liu; Deng; Timmons; Hersperger; Steeg; Veron; Shearn

    1996-08-01

    The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female

  3. Effects of point mutation on enzymatic activity: correlation between protein electronic structure and motion in chorismate mutase reaction.

    PubMed

    Ishida, Toyokazu

    2010-05-26

    Assignment of particular roles to catalytic residues is an important requirement in clearly understanding enzyme functions. Therefore, predicting the catalytic activities of mutant variants is a fundamental challenge in computational biochemistry. Although site-directed mutagenesis is widely used for studying enzymatic activities and other important classes of protein function, interpreting mutation experiments is usually difficult mainly due to side effects induced by point mutations. Because steric and, in many cases, electrostatic effects may affect the local, fine geometries conserved in wild-type proteins that are usually believed to be thermodynamically stable, simply reducing a loss in catalytic activity into clear elements is difficult. To address these important but difficult issues, we performed a systematic ab initio QM/MM computational analysis combined with MD-FEP simulations and all-electron QM calculations for the entire protein matrix. We selected chorismate mutase, one of the simplest and well-known enzymes, to discuss the details of mutational effects on the enzymatic reaction process. On the basis of the reliable free energy profiles of the wild-type enzyme and several mutant variants, we analyzed the effects of point mutations relative to electronic structure and protein dynamics. In general, changes in geometrical parameters introduced by a mutation were usually limited to the local mutational site. However, this local structural modification could affect the global protein dynamics through correlated motions of particular amino acid residues even far from the mutation site. Even for mutant reactions with low catalytic activity, transition state stabilization was observed as a result of conformational modifications and reorganization around the active site. As for the electrostatic effect created by the polar protein environment, the wild-type enzyme was most effectively designed to stabilize the transition state of the reactive substrate, and

  4. Longitudinal changes in PON1 enzymatic activities in Mexican-American mothers and children with different genotypes and haplotypes

    SciTech Connect

    Huen, Karen; Harley, Kim; Bradman, Asa; Eskenazi, Brenda; Holland, Nina

    2010-04-15

    The paraoxonase 1 (PON1) enzyme prevents low-density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican-American mothers and their children at the time of delivery (n = 388 and 338, respectively) and again 7 years later (n = 280 and 281, respectively) using generalized estimating equations models. At age 7, children's mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1{sub -108TT}, PON1{sub 192QQ}, and PON1{sub -909CC}). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to 7 years later when they were not pregnant (p < 0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, -108, -909, -162) explained a noticeably larger proportion of variance of paraoxonase activity (62-78%) than AREase activity (12.3-26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress.

  5. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    PubMed

    de Oliveira, Leandro C; da Silva, Viviam M; Colussi, Francieli; Cabral, Aline D; de Oliveira Neto, Mario; Squina, Fabio M; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

  6. Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

    PubMed Central

    de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  7. Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification.

    PubMed

    Vallance, Hilary; Morris, Tara J; Coulter-Mackie, Marion; Lim-Steele, Joyce; Kaback, Michael

    2006-02-01

    A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (alpha-subunit) and HEXB (beta-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher (P < 0.01) in subjects with low serum HexB enzymatic activities. Given the high frequency of the [delTG (+) 619A>G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

  8. Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.

    PubMed

    Kim, Ju Yaen; Kinoshita, Misaki; Kume, Satoshi; Gt, Hanke; Sugiki, Toshihiko; Ladbury, John E; Kojima, Chojiro; Ikegami, Takahisa; Kurisu, Genji; Goto, Yuji; Hase, Toshiharu; Lee, Young-Ho

    2016-11-01

    Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to (15)N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.

  9. The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

    PubMed

    Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

    2016-07-01

    In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m(-2)) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m(-2) dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m(-2) produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

  10. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    SciTech Connect

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  11. Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity.

    PubMed

    Baba, Reisuke; Hori, Yuichiro; Kikuchi, Kazuya

    2015-03-16

    Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

  12. Anti-diabetic activity of beta-glucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei.

    PubMed

    Kim, Yea-Woon; Kim, Ki-Hoon; Choi, Hyun-Ju; Lee, Dong-Seok

    2005-04-01

    Beta-glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of beta-glucans were 30-50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing beta-glucans with an endo-beta-(1-->6)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though beta-glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of beta-glucans with respect to anti-diabetic activity.

  13. A complete active space self-consistent field study of the photochemistry of nitrosamine

    NASA Astrophysics Data System (ADS)

    Peláez, Daniel; Arenas, Juan F.; Otero, Juan C.; Soto, Juan

    2006-10-01

    Photodissociation mechanisms of nitrosamine (NH2NO) have been studied at the complete active space self-consistent field level of theory in conjunction with atomic-natural-orbital-type basis sets. In addition, the energies of all the critical points and the potential energy curves connecting them have been recomputed with the multiconfigurational second-order perturbation method. Ground state minimum of nitrosamine has a C1 nonplanar structure with the hydrogen atoms of the amino moiety out of the plane defined by the N-N-O bonds. Electronic transitions to the three lowest states are allowed by selection rules: (i) S0→S3 (7.41eV) has an oscillator strength of f =0.0006 and it is assigned as an (npO)0→(πNO*)2 transition, (ii) S0→S2 (5.86eV ) has an oscillator strength of f =0.14 and it is assigned as an npN→πNO* transition, and (iii) S0→S1 (2.98eV ) has an oscillator strength of f =0.002 and it is assigned as an npO→πNO* transition. It is found that N-N bond cleavage is the most likely process in all the photochemical relevant states, namely, S1 (1A″1), S2 (2A'1), and T1 (1A″3). While S1 and T1 yield exclusively homolytic dissociation: NH2NO →NH2 (1B12)+NO(XΠ2), on S2 the latter process constitutes the major path, but two additional minor channels are also available: adiabatic homolytic dissociation: NH2NO →NH2 (1A12)+NO(XΠ2), and adiabatic oxygen extrusion: NH2NO →NH2N (1A13)+O(P3). The excited species NH2 (1A12) experiences a subsequent ultrafast decay to the ground state, the final products in all cases the fragments being in their lowest electronic state. We have not found a unimolecular mechanism connecting excited states with the ground state. In addition, homolytic dissociation in the ground state, tautomerizations to NHNOH and NHNHO, and intersystem crossings to T1 are considered. The most favorable process on this state is the isomerization to NHNOH.

  14. Seasonal Dynamics of Enzymatic Activities and Functional Diversity in Soils under Different Organic Management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil microbial activity and diversity fluctuate seasonally under annual organic amendment for improving soil quality. We investigated the effects of municipal compost (MC), poultry litter (PL), and cover crops of spring oats and red clover (RC) on soil enzyme activities, and soil bacterial community...

  15. Enzymatic characterizations and activity regulations of N-acetyl-β-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

    PubMed

    Zhang, Wei-Ni; Bai, Ding-Ping; Huang, Yi-Fan; Hu, Chong-Wei; Chen, Qing-Xi; Huang, Xiao-Hong

    2014-02-01

    N-Acetyl-β-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 μM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.

  16. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

    PubMed

    Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

    2015-03-01

    Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

  17. Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine.

    PubMed

    Abel, Biebele; Aslan, Kadir

    2012-01-01

    Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides.

  18. Methods, microfluidic devices, and systems for detection of an active enzymatic agent

    DOEpatents

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-10-28

    Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

  19. Influence of the isolation procedure on coriander leaf volatiles with some correlation to the enzymatic activity.

    PubMed

    To Quynh, Cung Thi; Iijima, Yoko; Kubota, Kikue

    2010-01-27

    Coriander leaves (Coriandrum sativum L.) have become popular worldwide because of their pleasant and delicate aroma. By a hot water extraction method, in which coriander leaves were cut before suspending in boiling water for 2 min, the contents of the main volatile compounds such as alkanals and 2-alkenals from C10 to C14 decreased, while the levels of corresponding alcohols increased in comparison to those obtained by solvent extraction. To investigate the reasons for this variation, an enzyme activity was assayed. By using aliphatic aldehyde as a substrate and NADPH as a coenzyme, strong activity of an aliphatic aldehyde reductase was found for the first time in this herb in the relatively wide pH range of 5.0-9.0, with the maximum activity at pH 8.5. Additionally, the aliphatic aldehyde dehydrogenase, responsible for acid formation, was also found to have a relatively weak activity compared to that of reductase.

  20. A methodology for preparing nanostructured biomolecular interfaces with high enzymatic activity

    NASA Astrophysics Data System (ADS)

    Wong, Lu Shin; Karthikeyan, Chinnan V.; Eichelsdoerfer, Daniel J.; Micklefield, Jason; Mirkin, Chad A.

    2012-01-01

    The development of a novel method for functionalizing nanopatterned surfaces with catalytically active proteins is reported. This method involves using dip-pen nanolithography (DPN) and polymer pen lithography (PPL) to generate nanoscale patterns of coenzyme A, followed by a phosphopantetheinyl transferase-mediated coupling between coenzyme A and proteins fused to the ybbR-tag. By exploiting the ability to generate protein features over large areas afforded by DPN and PPL, it was now possible to measure protein activity directly on these surfaces. It was found that proteins immobilized on the nanoscale features not only display higher activity per area with decreasing feature size, but are also robust and can be used for repeated catalytic cycles. The immobilization method is applicable to a variety of proteins and gives rise to superior activity compared to proteins attached in random orientations on the surface.

  1. Gene expression and enzymatic activity of pectin methylesterase during fruit development and ripening in Coffea arabica L.

    PubMed

    Cação, S M B; Leite, T F; Budzinski, I G F; dos Santos, T B; Scholz, M B S; Carpentieri-Pipolo, V; Domingues, D S; Vieira, L G E; Pereira, L F P

    2012-09-03

    Coffee quality is directly related to the harvest and post harvest conditions. Non-uniform maturation of coffee fruits, combined with inadequate harvest, negatively affects the final quality of the product. Pectin methylesterase (PME) plays an important role in fruit softening due to the hydrolysis of methylester groups in cell wall pectins. In order to characterize the changes occurring during coffee fruit maturation, the enzymatic activity of PME was measured during different stages of fruit ripening. PME activity progressively increased from the beginning of the ripening process to the cherry fruit stage. In silico analysis of expressed sequence tags of the Brazilian Coffee Genome Project database identified 5 isoforms of PME. We isolated and cloned a cDNA homolog of PME for further characterization. CaPME4 transcription was analyzed in pericarp, perisperm, and endosperm tissues during fruit development and ripening as well as in other plant tissues. Northern blot analysis revealed increased transcription of CaPME4 in the pericarp 300 days after flowering. Low levels of CaPME4 mRNAs were observed in the endosperm 270 days after flowering. Expression of CaPME4 transcripts was strong in the branches and lower in root and flower tissues. We showed that CaPME4 acts specifically during the later stages of fruit ripening and possibly contributes to the softening of coffee fruit, thus playing a significant role in pectin degradation in the fruit pericarp.

  2. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  3. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B.

    PubMed

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited.

  4. Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes.

    PubMed

    Jensen, Uffe B; Ferapontova, Elena E; Sutherland, Duncan S

    2012-07-31

    Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

  5. Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

    NASA Astrophysics Data System (ADS)

    Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

    2016-02-01

    We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

  6. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B

    PubMed Central

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M.; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  7. Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids.

    PubMed

    Fujita, Kyoko; Ohno, Hiroyuki

    2010-12-01

    Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp].

  8. Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase.

    PubMed

    Poe, R W; Sangadala, V S; Brewer, J M

    1993-05-15

    Seventeen salts were tested at various concentrations for their effects on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects. 1. All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M. The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations. These do not provide the secondary stimulatory effect of cations such as Na+ or Li+. 3. Anions associated with greater "salting in" effectiveness such as thiocyanate also reduce activity at ca. 1 M concentrations. These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation. There is little effect on the fluorescence or fluorescence-polarization spectrum of the enzyme so there is no general effect of 1 M salts on the conformation of the protein. The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax. Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.

  9. Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.

    PubMed

    Talipov, Marat R; Nayak, Jaladhi; Lepley, Michael; Bongard, Robert D; Sem, Daniel S; Ramchandran, Ramani; Rathore, Rajendra

    2016-11-08

    DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

  10. A complete active space self-consistent field study of the photochemistry of nitrosamine

    SciTech Connect

    Pelaez, Daniel; Arenas, Juan F.; Otero, Juan C.; Soto, Juan

    2006-10-28

    Photodissociation mechanisms of nitrosamine (NH{sub 2}NO) have been studied at the complete active space self-consistent field level of theory in conjunction with atomic-natural-orbital-type basis sets. In addition, the energies of all the critical points and the potential energy curves connecting them have been recomputed with the multiconfigurational second-order perturbation method. Ground state minimum of nitrosamine has a C{sub 1} nonplanar structure with the hydrogen atoms of the amino moiety out of the plane defined by the N-N-O bonds. Electronic transitions to the three lowest states are allowed by selection rules: (i) S{sub 0}{yields}S{sub 3} (7.41 eV) has an oscillator strength of f=0.0006 and it is assigned as an (np{sub O}){sup 0}{yields}({pi}{sub NO}*){sup 2} transition, (ii) S{sub 0}{yields}S{sub 2} (5.86 eV) has an oscillator strength of f=0.14 and it is assigned as an np{sub N}{yields}{pi}{sub NO}* transition, and (iii) S{sub 0}{yields}S{sub 1} (2.98 eV) has an oscillator strength of f=0.002 and it is assigned as an np{sub O}{yields}{pi}{sub NO}* transition. It is found that N-N bond cleavage is the most likely process in all the photochemical relevant states, namely, S{sub 1} (1 {sup 1}A{sup ''}), S{sub 2} (2 {sup 1}A{sup '}), and T{sub 1} (1 {sup 3}A{sup ''}). While S{sub 1} and T{sub 1} yield exclusively homolytic dissociation: NH{sub 2}NO{yields}NH{sub 2} (1 {sup 2}B{sub 1})+NO(X {sup 2}{pi}), on S{sub 2} the latter process constitutes the major path, but two additional minor channels are also available: adiabatic homolytic dissociation: NH{sub 2}NO{yields}NH{sub 2} (1 {sup 2}A{sub 1})+NO(X {sup 2}{pi}), and adiabatic oxygen extrusion: NH{sub 2}NO{yields}NH{sub 2}N (1 {sup 3}A{sub 1})+O({sup 3}P). The excited species NH{sub 2} (1 {sup 2}A{sub 1}) experiences a subsequent ultrafast decay to the ground state, the final products in all cases the fragments being in their lowest electronic state. We have not found a unimolecular mechanism connecting

  11. Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization.

    PubMed

    Orru, Roberto; Dudek, Hanna M; Martinoli, Christian; Torres Pazmiño, Daniel E; Royant, Antoine; Weik, Martin; Fraaije, Marco W; Mattevi, Andrea

    2011-08-19

    Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.

  12. Enzymatic activity of a mine soil varies according to vegetation cover and level of compost applied.

    PubMed

    de Varennes, Amerilis; Abreu, Maria Manuela; Qu, Guiwei; Cunha-Queda, Cristina

    2010-01-01

    We applied three doses of compost from mixed municipal solid waste (0, 15, and 30 g kg(-1) of soil) to a soil developed on pyrite mine wastes. Part of the soil was planted with young Erica australis L. collected at the mine; part was fertilized with N-P-K-Mg and sown with Dactylis glomerata L .Bare soil without mineral fertilization was included in the experiment, as well. Compost application to bare soil increased pH, provided plant nutrients, and enhanced the activity of the six soil enzymes tested. Growth of D. glomerata, and E. australis was stimulated in compost-amended soil compared with unamended controls. The presence of D. glomerata led to the greatest activities of soil acid phosphatase, beta-glucosidase, and cellulase compared with bare soil or with soil with E. australis. The presence of E. australis increased the activities of protease and cellulase in amended soil, compared with control, but it impaired dehydrogenase, fl-glucosidase, and acid phosphatase activities. These negative impacts probably derived from phenolic compounds known to be released from roots of this species. The survival strategy of this species seems to include a small need for P in the shoots, and the release of exudates that impair microbial activity and P cycling.

  13. Influence of Linker Length and Composition on Enzymatic Activity and Ribosomal Binding of Neomycin Dimers

    PubMed Central

    Watkins, Derrick; Kumar, Sunil; Green, Keith D.

    2015-01-01

    The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA. PMID:25896697

  14. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

    PubMed

    Boschin, Giovanna; Scigliuolo, Graziana Maria; Resta, Donatella; Arnoldi, Anna

    2014-02-15

    The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 μg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 μg/ml, was obtained hydrolysing a mixture of lupin α+β conglutin.

  15. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models

    PubMed Central

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q.

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  16. Functional and Behavioral Product Information Representation and Consistency Validation for Collaboration in Product Lifecycle Activities

    ERIC Educational Resources Information Center

    Baysal, Mehmet Murat

    2012-01-01

    Information models that represent the function, assembly and behavior of artifacts are critical in the conceptual development of a product and its evaluation. Much research has been conducted in this area; however, existing models do not relate function, behavior and structure in a comprehensive and consistent way. In this work, NIST's Core…

  17. Enzymatic activities in brains of diabetic rats treated with vanadyl sulphate and sodium tungstate.

    PubMed

    Lemberg, A; Fernández, M A; Ouviña, G; Rodríguez, R R; Peredo, H A; Susemihl, C; Villarreal, I; Filinger, E J

    2007-12-01

    The hypothesis of the present study was that diabetes mellitus might affect brain metabolism. Streptozotocin (STZ)-induced diabetic rats, treated with vanadyl sulphate (V) and sodium tungstate (T) were employed to observe the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) activities in brain homogenates. Significant increases in AST, ALT and CK activities were found in diabetic brain homogenates against controls, suggesting increments of transamination in brain and/or increases in cell membrane permeability to these enzymes. The increase in brain CK possibly expresses alterations in energy production. The decrease in CK activity caused by V and T treatment in diabetic rats suggests that both agents tend to normalize energy consumption. It is also possible that V and T-induced hypoglycemic effects cause metabolic alterations in brain.

  18. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  19. Long-term effects of nickel oxide nanoparticles on performance, microbial enzymatic activity, and microbial community of a sequencing batch reactor.

    PubMed

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Ma, Bingrui; Gao, Feng; Wang, Xuejiao

    2017-02-01

    The nitrogen and phosphorus removal, microbial enzymatic activity, and microbial community of a sequencing batch reactor (SBR) were evaluated under long-term exposure to nickel oxide nanoparticles (NiO NPs). High NiO NP concentration (over 5 mg L(-1)) affected the removal of chemical oxygen demand, nitrogen, and phosphorus. The presence of NiO NP inhibited the microbial enzymatic activities and reduced the nitrogen and phosphorus removal rates of activated sludge. The microbial enzymatic activities of the activated sludge showed a similar variation trend to the nitrogen and phosphorus removal rates with the increase in NiO NP concentration from 0 to 60 mg L(-1). The Ni content in the effluent and activated sludge showed an increasing trend with the increase in NiO NP concentration. Some NiO NPs were absorbed on the sludge surface or penetrate the cell membrane into the interior of microbial cells in the activated sludge. NiO NP facilitated the increase in reactive oxygen species by disturbing the balance between the oxidation and anti-oxidation processes, and the variation in lactate dehydrogenase demonstrated that NiO NP could destroy the cytomembrane and cause variations in the microbial morphology and physiological function. High-throughput sequencing demonstrated that the microbial community of SBR had some obvious changes at 0-60 mg L(-1) NiO NPs at the phyla, class and genus levels.

  20. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    PubMed Central

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    Summary We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence activated cell sorting. PMID:26060080

  1. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  2. Arid soil microbial enzymatic activity profile as affected by geographical location and soil degradation status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluating soil health is critical for any successful remediation effort. Arid lands, with their minimal carbon and water contents, low nutritional status and restricted, seasonal microbial activity pose specific challenges to soil health restoration and by extension, restoration of ecosystem repr...

  3. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

  4. Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.).

    PubMed

    Troncoso-Ponce, M Adrián; Garcés, Rafael; Martínez-Force, Enrique

    2010-12-01

    As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

  5. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    PubMed

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  6. Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1.

    PubMed

    Hachikubo, You; Ito, Kohji; Schiefelbein, John; Manstein, Dietmar J; Yamamoto, Keiichi

    2007-06-01

    We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V(max) and K(actin) of the actin-activated Mg(2+) ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 microM, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 microm s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 microm s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V(max) value of the actin-activated Mg(2+) ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.

  7. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties.

    PubMed

    Hedemann, M S; Eskildsen, M; Laerke, H N; Pedersen, C; Lindberg, J E; Laurinen, P; Knudsen, K E Bach

    2006-06-01

    The main objective of this study was to determine the effect of fiber source and concentration on morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8.6 +/- 1.4 kg) and divided into 5 treatment groups. Diets containing fiber of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of dietary fiber/kg of DM. The diets were based on raw wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration. The pigs were fed the experimental diets for 9 d, and then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken from the mid and distal small intestine and from the mid colon. Inclusion of pectin in the diets significantly decreased (P < 0.001) ADFI and ADG compared with pigs fed no pectin. The villi and the crypts were shorter in pigs fed pectin-containing diets, but the villous height/crypt depth ratio was unaltered. Pectin significantly decreased the area of mucins in the crypts of the small intestine, indicating that the pigs fed the pectin-containing diet would probably be more susceptible to pathogenic bacteria, although this cannot be separated from the impact on ADFI. The lectin-binding pattern of the intestinal mucosa was unaffected by diet. The activity of lactase and maltase was increased in pigs fed diets with high fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets. The activity of the peptidases, aminopeptidase N and dipeptidylpeptidase IV, was increased when feeding high fiber diets, whereas the activity of gamma-glutamyl transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain the

  8. Gamma irradiation effect on the enzymatic activities of horseradish and apple peroxidases

    NASA Astrophysics Data System (ADS)

    Constantinovici, M.; Oancea, D.; Zaharescu, T.

    2009-01-01

    The behavior at low-dose exposure (0.033-0.4 kGy) of horseradish peroxidase (HRP) and of two different purified fractions of apple (Jonathan cultivar) peroxidases (named APR 1S and APR 2S) was studied. The HRP solutions were added with either 0.32 M fructose or glucose in order to study their effect on enzymes activity response under γ ( 137Cs, dose rate 0.4 kGy/h) irradiation. The obtained results showed similar behavior between HRP-sugar-added solution and apple fraction with higher oligosaccharides content (APR 2S) undergoing low-dose treatment. The same pattern was observed between unglycosylated HRP and APR 1S with lower oligosaccharides content. These similarities gave us the possibility to conclude that the presence of oligosaccharides, in more or less quantities, influences in the same way the peroxidases activity, from different plant species, exposed to γ irradiation.

  9. Incorporating mobile nanospheres in the lumen of hybrid microcapsules for enhanced enzymatic activity.

    PubMed

    Shi, Jiafu; Zhang, Xiaoman; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

    2013-11-13

    Physical encapsulation of enzymes in microcapsules, as a mild, controllable method, has been widely utilized for enzyme immobilization. However, this method often suffers from the big mass transfer resistance from the capsule lumen. In this study, a novel biocatalysis system with enhanced catalytic activity is constructed through coencapsulating enzymes and nanospheres in the lumen of protamine/silica hybrid microcapsules, which are synthesized through the synergy of biomimetic silicification and layer-by-layer (LbL) assembly. When utilized as the host for catalase (CAT) encapsulation, the hybrid microcapsules maintain high mechanical stability, high enzyme loading, and low enzyme leaching. Particularly, because of the existence of mobile nanospheres, the mass transfer resistance in the microcapsules is significantly reduced because of the vigorous agitation, thus acquiring an enhanced catalytic activity. Our strategy may also find applications in drug delivery and biosensor fields.

  10. Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.

    PubMed

    Aragon, V; Kurtz, S; Flieger, A; Neumeister, B; Cianciotto, N P

    2000-04-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

  11. Probing Mechanisms for Enzymatic Activity Enhancement of Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun

    2009-12-25

    We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC - as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased a-helix/b-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

  12. Sulfonate derived phosphoramidates as active intermediates in the enzymatic primer-extension of DNA.

    PubMed

    De, S; Groaz, E; Margamuljana, L; Abramov, M; Marlière, P; Herdewijn, P

    2015-04-07

    Novel unnatural 5'-phosphoramidate nucleosides, capable of being processed as substrates by DNA polymerases for multiple nucleotide incorporations, have been designed. The mimics feature metabolites such as taurine and a broad range of aliphatic sulfonates coupled through a P-N bond to the 5'-phosphate position of deoxynucleotides, to allow binding interactions in the enzyme active site. The utility of all of the analogues as pyrophosphate mimics was demonstrated for the chain elongation of DNA, using both thermophilic and mesophilic microbial polymerases.

  13. Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

    PubMed Central

    Goettig, Peter

    2016-01-01

    Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. PMID:27898009

  14. Effect of high pressures on the enzymatic activity of commercial milk protein coagulants

    NASA Astrophysics Data System (ADS)

    Wiśniewska, Krystyna; Reps, Arnold; Jankowska, Agnieszka

    2014-04-01

    This study was aimed at determining the effect of high pressures in the range of 100-1000 MPa/15 min, applied in 100 MPa increments, on the coagulating and proteolytic activity of commercial coagulants produced with genetic engineering methods: Maxiren, Chymogen, Chymax and of a natural rennin preparation, Hala. The coagulating activity of Hala preparation differed compared with the other preparations, due to greater resistance to high pressures, especially in the range of 500-600 MPa. The preparations produced with genetic engineering methods lost their capability for milk protein coagulation by 500 MPa. Pressurization at 200 MPa contributed to their reduced capability for casein macroproteolysis. In contrast, an increase in Chymax, Chymogen, Maxiren and Hala preparations' hydrolytic capability for the macroproteolysis of isoelectric casein was observed upon pressure treatment at 100 and 400 MPa and for microproteolysis after pressure treatment at 200 MPa. Storage (48 h/5°C) of the pressurized preparations had an insignificant effect on their coagulating and proteolytic activities.

  15. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

    PubMed

    Verma, Shikha; Mehta, Ranjit Kumar; Maiti, Prasanta; Röhm, Klaus-Heinrich; Sonawane, Avinash

    2014-07-01

    Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.

  16. Antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced by enzymatic esterification.

    PubMed

    Vanin, Adriana B; Orlando, Tainara; Piazza, Suelen P; Puton, Bruna M S; Cansian, Rogério L; Oliveira, Debora; Paroul, Natalia

    2014-10-01

    This work reports the maximization of eugenyl acetate production by esterification of essential oil of clove in a solvent-free system using Novozym 435 as catalyst. The antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced were determined. The conditions that maximized eugenyl acetate production were 60 °C, essential oil of clove to acetic anhydride ratio of 1:5, 150 rpm, and 10 wt% of enzyme, with a conversion of 99.87 %. A kinetic study was performed to assess the influence of substrates' molar ratio, enzyme concentration, and temperature on product yield. Results show that an excess of anhydride, enzyme concentration of 5.5 wt%, 50 °C, and essential oil of clove to acetic anhydride ratio of 1:5 afforded nearly a complete conversion after 2 h of reaction. Comparing the antibacterial activity of the essential oil of clove before and after esterification, we observed a decrease in the antimicrobial activity of eugenyl acetate, particularly with regard to minimum inhibitory concentration (MIC). Both eugenyl acetate and clove essential oil were most effective to the gram-negative than gram-positive bacteria group. The results showed a high antioxidant potential for essential oil before and particularly after the esterification reaction thus becoming an option for the formulation of new antioxidant products.

  17. EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.

    PubMed

    Wehrman, Tom; Nguyen, Mimi; Feng, Wei; Bader, Benjamin

    2013-05-01

    Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.

  18. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity.

    PubMed

    Kaur, Gurvir; Tripathi, S K

    2015-01-05

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  19. Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress.

    PubMed

    Krueger, Thomas; Hawkins, Thomas D; Becker, Susanne; Pontasch, Stefanie; Dove, Sophie; Hoegh-Guldberg, Ove; Leggat, William; Fisher, Paul L; Davy, Simon K

    2015-12-01

    Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

  20. A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

    PubMed

    Pucher, Marion E; Steiger, Matthias G; Mach, Robert L; Mach-Aigner, Astrid R

    2011-06-10

    Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

  1. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity

    NASA Astrophysics Data System (ADS)

    Kaur, Gurvir; Tripathi, S. K.

    2015-01-01

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  2. Oil-containing waste water treating material consisting of modified active carbon

    SciTech Connect

    Sato, H.; Shigeta, S.; Takenaka, Y.

    1982-03-16

    An oil-containing waste water treating material comprises an active carbon upon whose surface is chemically bonded at least one nitrogenous compound which is an amine or a quaternarized derivative thereof.

  3. Biomechanics of the vibrissa motor plant in rat: rhythmic whisking consists of triphasic neuromuscular activity.

    PubMed

    Hill, Dan N; Bermejo, Roberto; Zeigler, H Philip; Kleinfeld, David

    2008-03-26

    The biomechanics of a motor plant constrain the behavioral strategies that an animal has available to extract information from its environment. We used the rat vibrissa system as a model for active sensing and determined the pattern of muscle activity that drives rhythmic exploratory whisking. Our approach made use of electromyography to measure the activation of all relevant muscles in both head-fixed and unrestrained rats and two-dimensional imaging to monitor the position of the vibrissae in head-fixed rats. Our essential finding is that the periodic motion of the vibrissae and mystacial pad during whisking results from three phases of muscle activity. First, the vibrissae are thrust forward as the rostral extrinsic muscle, musculus (m.) nasalis, contracts to pull the pad and initiate protraction. Second, late in protraction, the intrinsic muscles pivot the vibrissae farther forward. Third, retraction involves the cessation of m. nasalis and intrinsic muscle activity and the contraction of the caudal extrinsic muscles m. nasolabialis and m. maxillolabialis to pull the pad and the vibrissae backward. We developed a biomechanical model of the whisking motor plant that incorporates the measured muscular mechanics along with movement vectors observed from direct muscle stimulation in anesthetized rats. The results of simulations of the model quantify how the combination of extrinsic and intrinsic muscle activity leads to an enhanced range of vibrissa motion than would be available from the intrinsic muscles alone.

  4. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor.

    PubMed

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Ma, Bingrui; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Jin, Chunji; Wang, Xuejiao; Gao, Feng

    2017-02-01

    The long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial activity and microbial community of activated sludge were investigated in a sequencing batch reactor (SBR). The SBR performance had no evident change at 0-10 mg/L CuO NPs, whereas the CuO NPs concentration at 30-60 mg/L affected the COD, NH4(+)-N and soluble orthophosphate (SOP) removal, nitrogen and phosphorus removal rate and microbial enzymatic activity of activated sludge. Some CuO NPs might be absorbed on the surface of activated sludge or penetrate the microbial cytomembrane into the microbial cell interior of activated sludge. Compared to 0 mg/L CuO NPs, the reactive oxygen species (ROS) production and lactate dehydrogenase (LDH) release increased by 43.6% and 56.4% at 60 mg/L CuO NPs, respectively. The variations of ROS production and LDH release demonstrated that CuO NPs could induce the toxicity towards the microorganisms and destroy the integrity of microbial cytomembrane in the activated sludge. High throughput sequencing of 16S rDNA indicated that CuO NPs could evidently impact on the microbial richness, diversity and composition of activated sludge in the SBR.

  5. Relationships between microbial extracellular enzymatic activity and suspended and sinking particulate organic matter: seasonal transformations in the North Water

    NASA Astrophysics Data System (ADS)

    Huston, A. L.; Deming, J. W.

    Despite the importance of hydrolytic activities by bacterial extracellular enzymes (EE) in the temperate ocean, little is known about the role of extracellular enzymatic activity (EEA) in determining the fate of particulate organic matter (POM) in polar seas. To explore the issue further, we measured various chemical and bacterial parameters in the near-0°C waters of the North Water during the months of May and July of 1998. Seawater (SW) samples were collected by Niskin bottle at the depth of the chlorophyll fluorescence maximum (8-90 m), while samples of sinking particles and aggregates were collected in short-term (0.5-1.2 d), unpoisoned, floating sediment traps deployed at depths typically below the mixed layer (50-136 m). Samples were analyzed for POC, PON, and abundance of total and actively respiring bacteria. They were also incubated with fluorescently tagged substrate analogs to measure potential maximal rates of three classes of EE (leucine-aminopeptidase, chitobiase, and β-glucosidase) at -1°C. The percentage of actively respiring bacteria was always higher in sediment trap samples than in SW (medians of 38% and 24% versus 10% and 12% in May and July, respectively). Cell-specific rates of EEA were also higher in the trap samples and, for both sample types, similar to published rates from temperate waters. Rates of EEA when scaled to the abundance of actively respiring bacteria, however, did not differ between sample types, suggesting that the elevated EEA associated with sinking material is due to the greater abundance of metabolically active cells supported by such material and not due to enhanced enzyme expression in general, as suggested by previous studies. In this study, leucine-aminopeptidase activity was always much higher than the other classes of EEA, becoming even more dominant later in the season; it always correlated positively with the abundance of both total and actively respiring bacteria. Enzyme ratios indicating protease dominance

  6. The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei.

    PubMed

    Nalaskowski, Marcus M; Metzner, Anja; Brehm, Maria A; Labiadh, Sena; Brauer, Helena; Grabinski, Nicole; Mayr, Georg W; Jücker, Manfred

    2012-03-01

    The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

  7. Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

    PubMed

    Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E

    2010-03-01

    RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

  8. Induction of ferroxidase enzymatic activity by copper reduces MPP+-evoked neurotoxicity in rats.

    PubMed

    Rubio-Osornio, Moisés; Montes, Sergio; Heras-Romero, Yessica; Guevara, Jorge; Rubio, Carmen; Aguilera, Penélope; Rivera-Mancia, Susana; Floriano-Sánchez, Esaú; Monroy-Noyola, Antonio; Ríos, Camilo

    2013-03-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine, intracellular inclusions (Lewy bodies) and brain iron deposits. PD has also been related with reduced ferroxidase activity, diminished antioxidant defenses and lipid peroxidation. Striatal injection of 1-methyl-4-phenylpyridinium (MPP(+)) into rodents reproduces the major biochemical characteristics of PD, including oxidative stress. Copper (Cu) plays an important role as prosthetic group of several proteins involved in iron metabolism and antioxidant responses, such as ceruloplasmin (Cp). In the present study, intraperitoneal CuSO4 injection (10μmol/kg) produced an insignificant increase of Cu content in striatum and midbrain (17.5% and 7%, respectively). After 10 and 11h, Cu induced 6- and 4-fold increase Cp mRNA in midbrain and striatum, respectively. Cu-supplement also produced a time-dependent increase ferroxidase activity in striatal tissue, reaching a maximum 16h after Cu treatment in midbrain; while, ferrous iron content diminished 18% in striatum and 8% in midbrain. In regard the PD model, we found that MPP(+) (10μg/8μL, intrastriatal), induced a significant (P<0.05) reduction of striatal ferroxidase activity; this effect was reverted by Cu pre-treatment 16h before MPP(+). Likewise, Cu-supplement prevented lipid fluorescent products formation in striatum, evaluated (P<0.01) 6h after MPP(+). In the long term, apomorphine-evoked circling behavior was evaluated 6 days after MPP(+) injury; Cu pre-treatment significantly reduced (P<0.05) the apomorphine-induced ipsilateral turns in MPP(+)-lesioned rats. These results suggest that Cu-induced expression of Cp could be an interesting scope against the deleterious effects of iron deposits in PD.

  9. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  10. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

  11. Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

    PubMed Central

    Jo, Hakryul; Patterson, Victoria; Stoessel, Sean; Kuan, Chia-Yi; Hoh, Josephine

    2014-01-01

    High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development. PMID:25506911

  12. Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils.

    PubMed

    Selama, Okba; Amos, Gregory C A; Djenane, Zahia; Borsetto, Chiara; Laidi, Rabah Forar; Porter, David; Nateche, Farida; Wellington, Elizabeth M H; Hacène, Hocine

    2014-01-01

    Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  13. Lignin binding to pancreatic lipase and its influence on enzymatic activity.

    PubMed

    Zhang, Juan; Xiao, Lin; Yang, Yucai; Wang, Zhaoxia; Li, Genxi

    2014-04-15

    In this paper, we find that the effect of lignin on pancreatic lipase (PL) is dependent on reaction medium and substrate used. Experimental results reveal that lignin can gradually bind to PL to form a PL-lignin complex, resulting in an increased activity of the enzyme. The binding process is spontaneous and the PL-lignin complex formation is an endothermic reaction induced by hydrophobic and electrostatic interaction. There is a non-radiation energy transfer from PL to lignin during the binding process, and the binding of lignin to PL conforms to a secondary exponential decay function. Moreover, the α-helix content of the enzyme will be changed and the rigidity of its side chain will be enhanced due to the formation of lignin-PL complex. This study has not only provided the activation effect of lignin on PL, but also given an insight into the interaction between lignin and the enzyme, which would benefit the application of lignin in the pharmacy and food industry, as well as other fields.

  14. Cathepsin S of Sciaenops ocellatus: Identification, transcriptional expression and enzymatic activity.

    PubMed

    Sun, Bo-Guang; Chi, Heng

    2016-01-01

    Cathepsin S is a member of cysteine cathepsins and belongs to the cathepsin L-like family. In mammals, it is known to participate in various physiological processes and host immune defense. In teleost fish, the function of cathepsin S is less investigated. In the present work, we characterized a cathepsin S homologue (SoCatS) from red drum (Sciaenops ocellatus), a commercially valuable fish in Chinese mariculture. Like all cathepsin S, SoCatS possesses a peptidase domain with four catalytically essential residues (Gln140, Cys146, His285, and Asn305) conserved in the cathepsin S of different organisms. SoCatS shares 60-90% overall sequence identities with known teleost cathepsin S. Phylogenetic profiling indicated that SoCatS is evolutionally close to the cathepsin S of other teleost fish, especially Miichthys miiuy, a member of Sciaenidae family like red drum. SoCatS expression was detected in various tissues and was enhanced by bacterial infection. Purified recombinant SoCatS exhibited apparent peptidase activity with maximum at 50°C and pH 7.5. This activity depended on the catalytic residue Cys146 and was severely reduced by the cathepsin inhibitor E-64. Our results suggest that SoCatS functions as a cysteine protease which is probably involved in the antibacterial immunity of red drum.

  15. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  16. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

    PubMed Central

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O.; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  17. Changes in GDPase/UDPase enzymatic activity in response to oxidative stress in four Candida species.

    PubMed

    Delgado-Carmona, Jenny Daniela; Ramírez-Quijas, Mayra Denisse; Vega-González, Arturo; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2015-07-01

    The terminal processing of proteins and lipids occurs in the Golgi apparatus and involves the transport of sugar nucleotides into the Golgi lumen by specific carriers and the accumulation of nucleoside diphosphates (NDPs) as a result of oligosaccharide-protein glycosyltransferase activity. NDPs are converted into the corresponding nucleoside monophosphates (NMPs) by nucleoside diphosphatases (NDPases), thus relieving inhibition of sugar transferases. In addition, NMPs are then exchanged for equimolecular amounts of cytosolic sugar nucleotides by antiport transport systems. NDPases, commonly GDPase and UDPase, thus play a critical role in glycoprotein maturation and may influence fungal pathogenesis, morphogenesis, and cell wall properties. Interest of this laboratory has recently focused on the effect of reactive oxygen species (ROS) on enzymes involved in detoxification of these oxidants and on the metabolism of biomolecules such as lipids, nucleic acids, and proteins in human pathogenic Candida species. We therefore consider it important to extend these studies to determine how GDPase and UDPase are affected after exposure of cells to oxidants such as menadione, a superoxide (O2 (•-))-generator, and H2O2. Results indicate that activity of both enzymes decrease in response to these agents suggesting that ROS may also affect other critical cell functions such as protein glycosylation.

  18. The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization.

    PubMed

    Morettini, Stefano; Tribus, Martin; Zeilner, Anette; Sebald, Johanna; Campo-Fernandez, Beatriz; Scheran, Gabriele; Wörle, Hildegard; Podhraski, Valerie; Fyodorov, Dmitry V; Lusser, Alexandra

    2011-04-01

    The molecular motor protein CHD1 has been implicated in the regulation of transcription and in the transcription-independent genome-wide incorporation of H3.3 into paternal chromatin in Drosophila melanogaster. A key feature of CHD1 is the presence of two chromodomains, which can bind to histone H3 methylated at lysine 4 and thus might serve to recruit and/or maintain CHD1 at the chromatin. Here, we describe genetic and biochemical approaches to the study of the Drosophila CHD1 chromodomains. We found that overall localization of CHD1 on polytene chromosomes does not appreciably change in chromodomain-mutant flies. In contrast, the chromodomains are important for transcription-independent activities of CHD1 during early embryonic development as well as for transcriptional regulation of several heat shock genes. However, neither CHD1 nor its chromodomains are needed for RNA polymerase II localization and H3K4 methylation but loss of CHD1 decreases transcription-induced histone eviction at the Hsp70 gene in vivo. Chromodomain mutations negatively affect the chromatin assembly activities of CHD1 in vitro, and they appear to be involved in linking the ATP-dependent motor to the chromatin assembly function of CHD1.

  19. [The influence of smoking on plasma non-enzymatic antioxidant concentrations in active smokers (preliminary report)].

    PubMed

    Szołtysek-Bołdys, Izabela; Sobczak, Andrzej; Król, Dorota

    2006-01-01

    Tobacco smoke contains many reactive oxygen species (ROS) that cause oxidative stress. Crucial role in defending the organism against ROS play vitamins E and A. The aim of the study was to investigate the influence of tobacco smoke on concentration of main ingredients of these vitamins alpha-tocopherol and gamma-tocopherol, as well as retinol. The study population consisted of 104 healthy males between the age of 34 and 45 years. Survey questionnaire and determination of plasma cotinine concentration were used to divide the group into smokers (62 males) and non-smokers (42 males). The arbitrary threshold value of plasma cotinine concentration was set to 15 ng/ml. High performance liquid chromatography (HPLC) was used to estimate the plasma concentration of alpha- and gamma-tocopherol, retinol and cotinine. Within the smoking part of the study population a significantly lower (by 12.5%) concentration of alpha-tocopherol, and non-significantly higher (by 15.7%) concentration of gamma-tocopherol was ascertained, when compared to the plasma concentration of those compounds in the non-smoking group. Practically no difference in concentration of retinol was found between the two studied groups. In order to determine the magnitude of interdependency between the extensiveness of exposure to tobacco smoke and the concentration of analyzed antioxidants, correlations between their plasma concentrations and plasma concentration of cotinine were investigated. A significant, moderate and negative correlation of alpha-tocopherol versus cotinin was determined, in the smoking group as well as in the entire study population (r = -0.291 and r = - 0,317, respectively). Other relationship: gamma-tocopherol versus cotinine and retinol versus cotinine did not show any correlation. The obtained results suggest that tobacco smoke weakens the organism's antioxidant barrier by decreasing the concentration of plasma alpha-tocopherol, while not influencing significantly the plasma

  20. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2008-01-01

    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3

  1. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  2. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  3. Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Gandy, Michael N; Li, Jinghu; Zbytek, Blazej; Li, Wei; Tuckey, Robert C

    2005-08-01

    We demonstrate the metabolism of ergosterol by cytochrome P450scc in either a reconstituted system or isolated adrenal mitochondria. The major reaction product was identified as 17alpha,24-dihydroxyergosterol. Purified P450scc also generated hydroxyergosterol as a minor product, which is probably an intermediate in the synthesis of 17alpha,24-dihydroxyergosterol. In contrast to cholesterol and 7-dehydrocholesterol, cleavage of the ergosterol side chain was not observed. NMR analysis clearly located one hydroxyl group to C24, with evidence that the second hydroxyl group is at C17. 17alpha,24-Dihydroxyergosterol inhibited cell proliferation of HaCaT keratinocytes and melanoma cells. Thus, in comparison with cholesterol and 7-dehydrocholesterol, the 24-methyl group and the C22-C23 double bond of ergosterol prevent side chain cleavage by P450scc and change the enzyme's hydroxylase activity from C22 and C20, to C24 and C17, generating bioactive product.

  4. Dependence of Phospholipase D1 Multi-monoubiquitination on Its Enzymatic Activity and Palmitoylation*

    PubMed Central

    Yin, Hao; Gui, Yu; Du, Guangwei; Frohman, Michael A.; Zheng, Xi-Long

    2010-01-01

    Phospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1 H896R, K898R, or D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1 R691G,R695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrantly enlarged vesicles partially co-localized with the Golgi complex but not with early endosomes. The altered localization was reduced by the K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not of PLD1 K898R or PLD2, was blocked by inhibitors of proteasomes but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking between different membrane compartments. PMID:20189990

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

    PubMed

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

    2008-07-01

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

  6. Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products.

    PubMed

    Brinson, Robert G; Giulian, Gary G; Kelman, Zvi; Marino, John P

    2014-12-02

    Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.

  7. Rotation of nucleotide sites is not required for the enzymatic activity of chloroplast coupling factor

    SciTech Connect

    Musier, K.M.; Hammes, G.G.

    1987-09-22

    New heterobifunctional photoaffinity cross-linking reagents, 6-maleimido-N-(4-benzoylphenyl)hexanamide, 12-maleimido-N-(4-benzoylphenyl)dodecanamide, and 12-(/sup 14/C)maleimido-N-(4-benzoylphenyo)dodecanamide, were synthesized to investigate the mechanism of ATP hydrolysis by chloroplast coupling factor 1. These reagents react with sulfhydryl groups on the ..gamma..-polypeptide. Subsequent photolysis cross-links the ..gamma..-polypeptide covalently to ..cap alpha..- and ..beta..-polypeptides. The cross-linkers prevent major movements of the ..gamma..-polypeptide with respect to the ..cap alpha..- and ..beta..-polypeptides but are sufficiently long to permit some flexibility in the enzyme structure. When approx. 50% of the ..gamma..-polypeptide was cross-linked to a ..cap alpha..- and ..beta..-polypeptides, a 7% loss in ATPase activity was observed for the longer cross-linker and a 12% loss for the shorter. These results indicate that large movements of ..cap alpha..- and ..beta..-polypeptides with respect to the ..gamma..-polypeptide are not essential for catalysis. In particular, rotation of the polypeptide chains to crease structurally equivalent sites during catalysis is not a required feature of the enzyme mechanism.

  8. Toxicity of perfluorooctanoic acid towards earthworm and enzymatic activities in soil.

    PubMed

    He, Wenxiang; Megharaj, Mallavarapu; Naidu, Ravi

    2016-07-01

    Perfluorooctanoic acid (PFOA) is a widespread persistent organic contaminant in the environment that has recently raised much of regulatory and public concern. Therefore, assessment of its ecological risk is a top priority research. Hence, this study investigated the toxicity of PFOA to beneficial microbial processes in the soil such as activities of dehydrogenase, urease and potential nitrification in addition to earthworm survival, weight loss and PFOA bioaccumulation in two contrasting soils. In general, PFOA caused inhibition of all the measured microbial processes in a dose-dependent manner and the inhibition was higher in Williamtown (WT) soil than Edinburgh (EB) soil. Thus, WT soil being sandy in nature with low clay content showed higher PFOA bioavailability and hence showed higher toxicity. There was no mortality in earthworms exposed up to 100 mg PFOA/kilogram soil in both the soils; however, there was a significant weight loss from 25 mg/kg onwards. This study clearly demonstrates that soil contamination of PFOA can lead to adverse effects on soil health.

  9. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  10. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    PubMed Central

    Kristan, Katja; Deluca, Dominga; Adamski, Jerzy; Stojan, Jure; Rižner, Tea Lanišnik

    2005-01-01

    Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. PMID:16359545

  11. Consistent abnormalities in metabolic network activity in idiopathic rapid eye movement sleep behaviour disorder.

    PubMed

    Wu, Ping; Yu, Huan; Peng, Shichun; Dauvilliers, Yves; Wang, Jian; Ge, Jingjie; Zhang, Huiwei; Eidelberg, David; Ma, Yilong; Zuo, Chuantao

    2014-12-01

    Rapid eye movement sleep behaviour disorder has been evaluated using Parkinson's disease-related metabolic network. It is unknown whether this disorder is itself associated with a unique metabolic network. 18F-fluorodeoxyglucose positron emission tomography was performed in 21 patients (age 65.0±5.6 years) with idiopathic rapid eye movement sleep behaviour disorder and 21 age/gender-matched healthy control subjects (age 62.5±7.5 years) to identify a disease-related pattern and examine its evolution in 21 hemi-parkinsonian patients (age 62.6±5.0 years) and 16 moderate parkinsonian patients (age 56.9±12.2 years). We identified a rapid eye movement sleep behaviour disorder-related metabolic network characterized by increased activity in pons, thalamus, medial frontal and sensorimotor areas, hippocampus, supramarginal and inferior temporal gyri, and posterior cerebellum, with decreased activity in occipital and superior temporal regions. Compared to the healthy control subjects, network expressions were elevated (P<0.0001) in the patients with this disorder and in the parkinsonian cohorts but decreased with disease progression. Parkinson's disease-related network activity was also elevated (P<0.0001) in the patients with rapid eye movement sleep behaviour disorder but lower than in the hemi-parkinsonian cohort. Abnormal metabolic networks may provide markers of idiopathic rapid eye movement sleep behaviour disorder to identify those at higher risk to develop neurodegenerative parkinsonism.

  12. Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

    PubMed

    Müller, M; Schlapfer, B; Azzi, A

    1988-09-20

    Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

  13. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glasner, M.; Vick, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2007-01-01

    The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain and a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only

  14. Extraction and quantitation of coumarin from cinnamon and its effect on enzymatic browning in fresh apple juice: a bioinformatics approach to illuminate its antibrowning activity.

    PubMed

    Thada, Rajarajeshwari; Chockalingam, Shivashri; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar

    2013-06-05

    Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice.

  15. 1-(Fluoroalkylidene)-1,1-bisphosphonic Acids are Potent and Selective Inhibitors of the Enzymatic Activity of Toxoplasma gondii Farnesyl Pyrophosphate Synthase

    PubMed Central

    Szajnman, Sergio H.; Rosso, Valeria S.; Malayil, Leena; Smith, Alyssa; Moreno, Silvia N. J.; Docampo, Roberto

    2012-01-01

    α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease and against Toxoplasma gondii, the responsible agent of toxoplasmosis and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) has proven to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range exhibiting an IC50 of 30 nM. This compound was two-fold more potent than risedronate (IC50 = 74 nM) taken as a positive control. This enzymatic activity was associated to a strong cell growth inhibition against tachyzoites of T. gondii having an IC50 value of 2.7 μM. PMID:22215028

  16. alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress.

    PubMed

    Melkani, Girish C; Cammarato, Anthony; Bernstein, Sanford I

    2006-05-05

    Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis

  17. Genome-Wide Identification, 3D Modeling, Expression and Enzymatic Activity Analysis of Cell Wall Invertase Gene Family from Cassava (Manihot esculenta Crantz)

    PubMed Central

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun

    2014-01-01

    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker. PMID:24786092

  18. Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

    PubMed

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun

    2014-04-28

    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

  19. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation.

    PubMed

    Walcott, Sam; Kad, Neil M

    2015-11-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies.

  20. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  1. Teaching hands-on science activities: Variables that moderate attitude-behavior consistency

    NASA Astrophysics Data System (ADS)

    Koballa, Thomas R., Jr.

    The relationship between prospective elementary teachers' attitudes, subjective norms, and intentions to teach science using hands-on activities at least twice a week during their first year of employment was investigated. The findings suggest that measuring prospective teachers' attitudes toward science cannot adequately predict nor provide a satisfactory explanation of their science teaching behaviors. The findings also provide clear support for two hypotheses derived from Fishbein and Ajzen's theory of reasoned action regarding the predictability of prospective teachers' intentions to teach science from their attitudes and subjective norms.Received: 25 October 1985

  2. [Some enzymatic activities of the amniotic fluid in human beings (LAP, GGTP, SGOT, SGPT, acid and alkaline phosphatases, 5' nucleotidase, amylase, beta-glucuronidase and aldolase)].

    PubMed

    Galerne, D; Baudon, J; Bruhat, M; Dastugue, G

    1973-10-01

    Quantitative analyses of 10 enzymes (LAP, GGTP, SGOT, SGPT. acid and alkaline phosphatases, 5' nucleotidase, amylase. beta-glucuronidase and aldolase) in a series of 50 samples of amniotic fluid gave widely-scattered results. In some cases, it was possible to relate high enzymatic activity to a pathological condition, in other cases, the amniotic fluid examined seemed to come from normal, full-term or almost full-term pregnancies without particular signs.

  3. Enzymatic activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and exploration types in an afrotropical rain forest.

    PubMed

    Tedersoo, Leho; Naadel, Triin; Bahram, Mohammad; Pritsch, Karin; Buegger, Franz; Leal, Miguel; Kõljalg, Urmas; Põldmaa, Kadri

    2012-09-01

    Ectomycorrhizal (ECM) fungi obtain both mineral and simple organic nutrients from soil and transport these to plant roots. Natural abundance of stable isotopes (¹⁵N and ¹³C) in fruit bodies and potential enzymatic activities of ECM root tips provide insights into mineral nutrition of these mutualistic partners. By combining rDNA sequence analysis with enzymatic and stable isotope assays of root tips, we hypothesized that phylogenetic affinities of ECM fungi are more important than ECM exploration type, soil horizon and host plant in explaining the differences in mineral nutrition of trees in an African lowland rainforest. Ectomycorrhizal fungal species belonging to extraradical mycelium-rich morphotypes generally displayed the strongest potential activities of degradation enzymes, except for laccase. The signature of ¹⁵N was determined by the ECM fungal lineage, but not by the exploration type. Potential enzymatic activities of root tips were unrelated to ¹⁵N signature of ECM root tip. The lack of correlation suggests that these methods address different aspects in plant nutrient uptake. Stable isotope analysis of root tips could provide an additional indirect assessment of fungal and plant nutrition that enables enhancement of taxonomic coverage and control for soil depth and internal nitrogen cycling in fungal tissues.

  4. The effect of Eulaliopsis binata on the physi-chemical properties, microbial biomass, and enzymatic activities in Cd-Pb polluted soil.

    PubMed

    Yu, Hui; Xiang, Yanci; Zou, Dongsheng

    2016-10-01

    Pot culture experiment using mining wasteland soil was carried out to study the effect of Eulaliopsis binata on the heavy-metal polluted soil with the growth of 90, 180, 270, and 360 days. Soil nutritional components, heavy metal, microbial biomass, and enzymatic activities were analyzed in this study, and the control group had no plants. The results showed that heavy metal contents decreased with E. binata growth, extractable Cd and Pb decreased 28 and 15 % after 1 year, but the difference was not significant compared with the control. While soil nutritional components, microbial biomass and enzymatic activities increased significantly as compared with the control. Comparing with pre-experiment, soil organic matter, N, P, K, microbial biomass C, N, P, invertase, urease, acid phosphatase, and catalase increased 0.9, 1.1, 3.0, 1.1, 0.4, 0.3, and 0.5 times, respectively. The indexes of soil nutritional components, microbial biomass, and enzymatic activities are positively correlated to each other, while they are negatively correlated to heavy metal content respectively. E. binata has positive influence on Cd-Pb pollution soil and broad application prospects in remediating heavy-metal polluted soil.

  5. Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase.

    PubMed

    Kawazu, T; Nakanishi, Y; Uozumi, N; Sasaki, T; Yamagata, H; Tsukagoshi, N; Udaka, S

    1987-04-01

    The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis. Homologous DNA was found by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end. The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods. It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time. The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases.

  6. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

  7. Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

    PubMed

    Otsuka, Yuichiro; Matsuda, Motoki; Sonoki, Tomonori; Sato-Izawa, Kanna; Goodell, Barry; Jelison, Jody; Navarro, Ronald R; Murata, Hitoshi; Nakamura, Masaya

    2016-12-01

    This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

  8. Altering enzymatic activity: recruitment of carboxypeptidase activity into an RTEM beta-lactamase/penicillin-binding protein 5 chimera.

    PubMed

    Chang, Y H; Labgold, M R; Richards, J H

    1990-04-01

    The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A beta-lactamases (EC 3.5.2.6), although these beta-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a beta-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 beta-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during beta-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM beta-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A beta-lactamases and are thought to be essential for beta-lactamase activity, the chimera has considerably reduced activity (approximately 10(-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of approximately 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme.

  9. Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity.

    PubMed

    Shiman, R; Xia, T; Hill, M A; Gray, D W

    1994-10-07

    Activation by phenylalanine and reduction by the co-factor (6R)-tetrahydrobiopterin (BH4) are required for formation of active liver phenylalanine hydroxylase. This work describes effects of the activation and redox state on substrate and effector recognition of this enzyme, it establishes relationships among the pterin and phenylalanine binding sites on the different forms of the enzyme, and it provides a quantitative description of the enzyme's presumptive regulatory and catalytic sites. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-deaza-6-methyltetrahydropterin were found to bind to unactivated phenylalanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site on the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. In the transition from unactivated to activated enzyme, phenylalanine and pterin binding is modified, a new site for phenylalanine is formed, and the pterin site is replaced by a site of greatly decreased affinity for BH4 and BH2, one which does not appear to recognize the dihydroxypropyl side chain of BH4 and BH2. The pterin- and phenylalanine-binding sites on activated phenylalanine hydroxylase appear to be part of the enzyme's active site. Despite large effects on substrate binding, neither chelator binding ability nor solvent accessibility of the iron are affected by activation; activation appears to affect the nearby environment of the enzyme's iron but not the iron itself. Studies of oxidized and reduced phenylalanine hydroxylase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.

  10. 1H, 13C, and 15N resonance assignments of an enzymatically active domain from the catalytic component (CDTa, residues 216-420) of a binary toxin from Clostridium difficile.

    PubMed

    Roth, Braden M; Godoy-Ruiz, Raquel; Varney, Kristen M; Rustandi, Richard R; Weber, David J

    2016-04-01

    Clostridium difficile is a bacterial pathogen and is the most commonly reported source of nosocomial infection in industrialized nations. Symptoms of C. difficile infection (CDI) include antibiotic-associated diarrhea, pseudomembranous colitis, sepsis and death. Over the last decade, rates and severity of hospital infections in North America and Europe have increased dramatically and correlate with the emergence of a hypervirulent strain of C. difficile characterized by the presence of a binary toxin, CDT (C. difficile toxin). The binary toxin consists of an enzymatic component (CDTa) and a cellular binding component (CDTb) that together form the active binary toxin complex. CDTa harbors a pair of structurally similar but functionally distinct domains, an N-terminal domain (residues 1-215; (1-215)CDTa) that interacts with CDTb and a C-terminal domain (residues 216-420; (216-420)CDTa) that harbors the intact ADP-ribosyltransferase (ART) active site. Reported here are the (1)H, (13)C, and (15)N backbone resonance assignments of the 23 kDa, 205 amino acid C-terminal enzymatic domain of CDTa, termed (216-420)CDTa. These NMR resonance assignments for (216-420)CDTa represent the first for a family of ART binary toxins and provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

  11. Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

    PubMed

    Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

    2015-06-01

    An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g.

  12. Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

    NASA Astrophysics Data System (ADS)

    Han, Wenjun; Gu, Jingyan; Liu, Huihui; Li, Fuchuan; Wu, Zhihong; Li, Yuezhong

    2015-10-01

    A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo- β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

  13. pCO2 and enzymatic activity in a river floodplain system of the Danube under different hydrological settings.

    NASA Astrophysics Data System (ADS)

    Sieczko, Anna; Demeter, Katalin; Mayr, Magdalena; Meisterl, Karin; Peduzzi, Peter

    2014-05-01

    Surface waters may serve as either sinks or sources of CO2. In contrast to rivers, which are typically sources of CO2 to the atmosphere, the role of fringing floodplains in CO2 flux is largely understudied. This study was conducted in a river-floodplain system near Vienna (Austria). The sampling focused on changing hydrological situations, particularly on two distinct flood events: a typical 1-year flood in 2012 and an extraordinary 100-year flood in 2013. One objective was to determine partial pressure of CO2 (pCO2) in floodplain lakes with different degree of connectivity to the main channel, and compare the impact of these two types of floods. Another aim was to decipher which fraction of the dissolved organic matter (DOM) pool contributed to pCO2 by linking pCO2 with optical properties of DOM and extracellular enzymatic activity (EEA) of microbes. The EEA is a valuable tool, especially for assessing the non-chromophoric but rapidly utilized DOM-fraction during floods. In 2012 and 2013, the floodplain lakes were dominated by supersaturated pCO2 conditions, which indicates that they served as CO2 sources. Surprisingly, there were no significant differences in pCO2 between the two types of flood. Our findings imply that the extent of the flood had minor impact on pCO2, but the general occurrence of a flood appears to be important. During the flood in 2013 significantly more dissolved organic carbon (DOC) (p<0.05) was introduced into the floodplain. The optical measurements pointed towards more refractory DOM, with higher molecular weight and humic content during the flood in 2013 compared to 2012. However there were no significant differences in EEA between the two floods. Few days after beginning of the floods in 2012 and 2013, an increase in activity of carbon-acquiring enzymes (EEA-C) was observed. We also found positive correlations of pCO2with EEA-C both in 2012 (r=0.86, p<0.01) and in 2013 (r=0.73, p<0.05). The above findings imply that some fraction of DOM

  14. Enzymatic vitreous disruption.

    PubMed

    Gandorfer, A

    2008-10-01

    Enzymatic vitreous disruption refers to cleaving the vitreoretinal junction by enzymatic means, thereby inducing posterior vitreous detachment (PVD) and liquefaction of the vitreous gel. Several enzymes have been proposed in this respect, including chondroitinase, hyaluronidase, dispase, and plasmin. In an experimental setting, chondroitinase induced PVD and was helpful in removing epiretinal membranes but no further data have been reported yet. Hyaluronidase liquefies the vitreous as demonstrated in a phase III trial in diabetic patients with vitreous haemorrhage. Dispase induces PVD but also causes inner retinal damage and is now used as an animal model of proliferative vitreoretinopathy. Plasmin has the capability of both PVD induction and liquefaction. However, plasmin is highly unstable and not available for clinical use. Microplasmin (ThromboGenics Ltd, Dublin, Ireland) is a truncated form of human plasmin sharing the same catalytic activity like plasmin. Recombinant microplasmin is under clinical investigation in patients with vitreomacular traction. This review article reports on the current knowledge of enzymatic vitreous disruption and discusses details of the enzyme candidates in basic and clinical research terms.

  15. On the enzymatic activity of catalase: an iron L-edge X-ray absorption study of the active centre.

    PubMed

    Bergmann, Nora; Bonhommeau, Sébastien; Lange, Kathrin M; Greil, Stefanie M; Eisebitt, Stefan; de Groot, Frank; Chergui, Majed; Aziz, Emad F

    2010-05-14

    Catalase and methaemoglobin have very similar haem groups, which are both ferric, yet catalase decomposes hydrogen peroxide to water and oxygen very efficiently, while methaemoglobin does not. Structural studies have attributed this behaviour to their different distal environments. Here we present Fe L(2,3)-edge X-ray absorption spectra of these proteins in physiological solutions, which reveal clear differences in their electronic structures, in that pi back-donation of the Fe atom occurs in catalase, which confers on it a partial ferryl (Fe(4+)) character, while this is not the case in methaemoglobin. The origin of the Fe(4+) character stems from the proximal tyrosine residue. We also find that both systems are in a high spin state. Temperature effects influence the spectra of catalase only weakly, in agreement with previous studies of its chemical activity. We conclude that the high activity of catalase is not only determined by its distal environment but also by its partial ferryl character.

  16. Optimizing the enzymatic synthesis of beta-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity in vivo.

    PubMed

    Hermida, Carmen; Corrales, Guillermo; Cañada, Francisco J; Aragón, Juan J; Fernández-Mayoralas, Alfonso

    2007-07-15

    Disaccharides 2-O-, 3-O-, and 4-O-beta-D-galactopyranosyl-D-xyloses (2, 3, and 1, respectively) were obtained by beta-galactosidase-catalyzed reactions for their use in the evaluation of intestinal lactase activity in vivo. Their administration to suckling rats followed by determination of the derived D-xylose in the urine and measurement of lactase activity in intestinal homogenates showed 1 to be the most suitable disaccharide for a potential test of the deficiency of intestinal lactase. The synthesis of 1 was further studied by evaluating the effect of different variables on the yield and regioselectivity of the enzymatic galactosylation, and the purification process was optimized.

  17. Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

    PubMed

    Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

    2013-09-15

    Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes.

  18. Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates.

    PubMed

    Liu, Hai-Jing; Tang, Zhen-Xin; Han, Xue-Min; Yang, Zhi-Ling; Zhang, Fu-Min; Yang, Hai-Ling; Liu, Yan-Jing; Zeng, Qing-Yin

    2015-11-01

    Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.

  19. Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

    PubMed

    Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

    2015-02-01

    The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

  20. Identification of the Regions of Cytotoxic Necrotizing Factor Type 1 Responsible for Receptor Binding and Enzymatic Activity

    DTIC Science & Technology

    2007-02-05

    produce two toxins , hemolysin and CNF1, as well as other virulence determinants such as lipopolysaccharide (LPS) and iron acquisition products (not shown...cytoplasmic polypeptide toxin that is composed of a reputed N-terminal binding domain and a C- terminal enzymatic domain. A putative transmembrane...domain, considered to be responsible for translocation of the toxin into eukaryotic cells, is contained within the N- terminal half of the molecule

  1. Human procaspase-1 variants with decreased enzymatic activity are associated with febrile episodes and may contribute to inflammation via RIP2 and NF-κB signaling.

    PubMed

    Heymann, Michael C; Winkler, Stefan; Luksch, Hella; Flecks, Silvana; Franke, Marcus; Ruß, Susanne; Ozen, Seza; Yilmaz, Engin; Klein, Christoph; Kallinich, Tilmann; Lindemann, Dirk; Brenner, Sebastian; Ganser, Gerd; Roesler, Joachim; Rösen-Wolff, Angela; Hofmann, Sigrun R

    2014-05-01

    The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

  2. Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity.

    PubMed

    Calvez, Philippe; Breukink, Eefjan; Roper, David I; Dib, Mélanie; Contreras-Martel, Carlos; Zapun, André

    2017-02-17

    Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism.

  3. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  4. Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein

    PubMed Central

    2013-01-01

    Background Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). Results Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. Conclusions Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE). PMID:23448268

  5. A sensitive radioisotopic method for the measurement of NAD(P)H: Its application to the assay of metabolites and enzymatic activities

    SciTech Connect

    Sener, A.; Malaisse, W.J. )

    1990-05-01

    A radioisotopic method for the assay of NADH or NADPH is presented, which is based on the conversion of 2-(U-{sup 14}C)ketoglutarate to {sup 14}C-labeled glutamate in the reaction catalyzed by glutamate dehydrogenase. The efficiency of the method is close to 75%, its precision (coefficient of variation) close to 5%, and its sensitivity close to 0.1 pmol/sample. This simple and rapid method can be applied to the measurement of several metabolites and enzymatic activities. In the present study, its application to the assay of sorbitol, 3-hydroxybutyrate, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase is documented.

  6. Fully relativistic complete active space self-consistent field for large molecules: Quasi-second-order minimax optimization

    SciTech Connect

    Bates, Jefferson E.; Shiozaki, Toru

    2015-01-28

    We develop an efficient algorithm for four-component complete active space self-consistent field (CASSCF) methods on the basis of the Dirac equation that takes into account spin–orbit and other relativistic effects self-consistently. Orbitals are optimized using a trust-region quasi-Newton method with Hessian updates so that energies are minimized with respect to rotations among electronic orbitals and maximized with respect to rotations between electronic and positronic orbitals. Utilizing density fitting and parallel computation, we demonstrate that Dirac–Coulomb CASSCF calculations can be routinely performed on systems with 100 atoms and a few heavy-elements. The convergence behavior and wall times for octachloridodirhenate(III) and a tungsten methylidene complex are presented. In addition, the excitation energies of octachloridodirhenate(III) are reported using a state-averaged variant.

  7. Fully relativistic complete active space self-consistent field for large molecules: Quasi-second-order minimax optimization

    NASA Astrophysics Data System (ADS)

    Bates, Jefferson E.; Shiozaki, Toru

    2015-01-01

    We develop an efficient algorithm for four-component complete active space self-consistent field (CASSCF) methods on the basis of the Dirac equation that takes into account spin-orbit and other relativistic effects self-consistently. Orbitals are optimized using a trust-region quasi-Newton method with Hessian updates so that energies are minimized with respect to rotations among electronic orbitals and maximized with respect to rotations between electronic and positronic orbitals. Utilizing density fitting and parallel computation, we demonstrate that Dirac-Coulomb CASSCF calculations can be routinely performed on systems with 100 atoms and a few heavy-elements. The convergence behavior and wall times for octachloridodirhenate(III) and a tungsten methylidene complex are presented. In addition, the excitation energies of octachloridodirhenate(III) are reported using a state-averaged variant.

  8. Fully relativistic complete active space self-consistent field for large molecules: quasi-second-order minimax optimization.

    PubMed

    Bates, Jefferson E; Shiozaki, Toru

    2015-01-28

    We develop an efficient algorithm for four-component complete active space self-consistent field (CASSCF) methods on the basis of the Dirac equation that takes into account spin-orbit and other relativistic effects self-consistently. Orbitals are optimized using a trust-region quasi-Newton method with Hessian updates so that energies are minimized with respect to rotations among electronic orbitals and maximized with respect to rotations between electronic and positronic orbitals. Utilizing density fitting and parallel computation, we demonstrate that Dirac-Coulomb CASSCF calculations can be routinely performed on systems with 100 atoms and a few heavy-elements. The convergence behavior and wall times for octachloridodirhenate(III) and a tungsten methylidene complex are presented. In addition, the excitation energies of octachloridodirhenate(III) are reported using a state-averaged variant.

  9. Major Protein of Resting Rhizomes of Calystegia sepium (Hedge Bindweed) Closely Resembles Plant RNases But Has No Enzymatic Activity1

    PubMed Central

    Van Damme, Els J.M.; Hao, Qiang; Barre, Annick; Rougé, Pierre; Van Leuven, Fred; Peumans, Willy J.

    2000-01-01

    The most abundant protein of resting rhizomes of Calystegia sepium (L.) R.Br. (hedge bindweed) has been isolated and its corresponding cDNA cloned. The native protein consists of a single polypeptide of 212 amino acid residues and occurs as a mixture of glycosylated and unglycosylated isoforms. Both forms are derived from the same preproprotein containing a signal peptide and a C-terminal propeptide. Analysis of the deduced amino acid sequence indicated that the C. sepium protein shows high sequence identity and structural similarity with plant RNases. However, no RNase activity could be detected in highly purified preparations of the protein. This apparent lack of activity results most probably from the replacement of a conserved His residue, which is essential for the catalytic activity of plant RNases. Our findings not only demonstrate the occurrence of a catalytically inactive variant of an S-like RNase, but also provide further evidence that genes encoding storage proteins may have evolved from genes encoding enzymes or other biologically active proteins. PMID:10677436

  10. Enzymatic degradation products from a marine polysaccharide YCP with different immunological activity and binding affinity to macrophages, hydrolyzed by alpha-amylases from different origins.

    PubMed

    Ren, Min; Yan, Wei; Yao, Wenbing; Jin, Lei; Gao, Xiangdong

    2010-04-01

    YCP is a marine polysaccharide with anti-tumor and immune-modulating effects. This study evaluated the effect of enzymatic degradation of YCP by alpha-amylases from different origins on its immunological activity and binding ability to the macrophages. YCP was hydrolyzed by alpha-amylases isolated from Aspergillus oryzae, Bacillus licheniformis, Barley malt, and Porcine pancreas respectively, then four fragments with unique molecular weight (termed: YCP-Ao, YCP-Bl, YCP-Bm, and YCP-Pp, respectively) were obtained. The four fragments showed different immunological activity and the ability to bind to macrophages. Among them, YCP-Ao possessed almost equivalent immunological activity compared to the original YCP, while such properties were not retained in YCP-Bl. Our further study showed that YCP-Ao prevented YCP from binding to macrophages. In conclusion, YCP-Ao and YCP might have similar active regions.

  11. Oxygen isotope ratios of PO4: an inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life.

    PubMed

    Blake, R E; Alt, J C; Martini, A M

    2001-02-27

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (delta(18)O(p)) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO(4)-H(2)O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that delta(18)O(P) values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of delta(18)O(p) as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, delta(18)O(p) may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

  12. Special Feature: Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

    NASA Astrophysics Data System (ADS)

    Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

    2001-02-01

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity,the demonstration of enzyme-catalyzed PO4-H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

  13. Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

    PubMed Central

    Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

    2001-01-01

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars. PMID:11226207

  14. Perturbative correction for the basis set incompleteness error of complete-active-space self-consistent field.

    PubMed

    Kong, Liguo; Valeev, Edward F

    2010-11-07

    To reduce the basis set incompleteness of the complete-active-space self-consistent field (CASSCF) wave function and energy we develop a second-order perturbation correction due to single excitations to complete set of unoccupied states. Other than the one- and two-electron integrals, only one- and two-particle reduced density matrices are required to compute the correction, denoted as [2](S). Benchmark calculations on prototypical ground-state bond-breaking problems show that only the aug-cc-pVXZ basis is needed with the [2](S) correction to match the accuracy of CASSCF energies of the aug-cc-pV(X+1)Z quality.

  15. An atomic orbital-based formulation of the complete active space self-consistent field method on graphical processing units

    SciTech Connect

    Hohenstein, Edward G.; Luehr, Nathan; Ufimtsev, Ivan S.; Martínez, Todd J.

    2015-06-14

    Despite its importance, state-of-the-art algorithms for performing complete active space self-consistent field (CASSCF) computations have lagged far behind those for single reference methods. We develop an algorithm for the CASSCF orbital optimization that uses sparsity in the atomic orbital (AO) basis set to increase the applicability of CASSCF. Our implementation of this algorithm uses graphical processing units (GPUs) and has allowed us to perform CASSCF computations on molecular systems containing more than one thousand atoms. Additionally, we have implemented analytic gradients of the CASSCF energy; the gradients also benefit from GPU acceleration as well as sparsity in the AO basis.

  16. Structure, enzymatic transformation and anticancer activity of branched high molecular weight laminaran from brown alga Eisenia bicyclis.

    PubMed

    Menshova, Roza V; Ermakova, Svetlana P; Anastyuk, Stanislav D; Isakov, Vladimir V; Dubrovskaya, Yuliya V; Kusaykin, Mikhail I; Um, Byung-Hun; Zvyagintseva, Tatiana N

    2014-01-01

    The structure of high molecular weight laminaran from brown alga Eisenia bicyclis was investigated by chemical and enzymatic methods, NMR spectroscopy and mass spectrometry. The laminaran from E. bicyclis was characterized as 1,3;1,6-β-D-glucan with the high content of 1,6-linked glucose residues (ratio of bonds 1,3:1,6=1.5:1), which are both in the branches and in the main chain of the laminaran. The degree of polymerization of fragments, building from 1,3-linked glucose residues with single glucose branches at C-6 or without it, was no more than four glucose residues. The main part of 1,3-linked glucose blocks was builded from disaccharide fragments. 1,6-Linked glucose residues were localized basically on non-reduced ends of molecules. The degree of polymerization of 1,6-linked blocks was not greater than three glucose residues. Laminaran contained laminarioligosaccharides, gentiobiose, gentiotriose and single glucose residues in the branches at the C-6. Laminaran and its products of enzymatic hydrolysis inhibited a colony formation of human melanoma SK-MEL-28 and colon cancer DLD-1 cells. It was shown that decreasing the molecular weight of native laminaran to a determined limit (degree of polymerization 9-23) and increasing the content of 1,6-linked glucose residues increased the anticancer effect. Therefore, they may be perspective antitumor agents.

  17. Enzymatic Regulation of Self-Assembling Peptide A9K2 Nanostructures and Hydrogelation with Highly Selective Antibacterial Activities.

    PubMed

    Bai, Jingkun; Chen, Cuixia; Wang, Jingxin; Zhang, Yu; Cox, Henry; Zhang, Jing; Wang, Yuming; Penny, Jeffrey; Waigh, Thomas; Lu, Jian R; Xu, Hai

    2016-06-22

    Hydrogels offer great potential for many biomedical and technological applications. For clinical uses, hydrogels that act as scaffold materials for cell culture, regenerative medicine, and drug delivery are required to have bactericidal properties. The amphiphilic peptide A9K2 was designed to effectively inhibit bacterial growth via a mechanism of membrane permeabilization. The present study demonstrated that addition of fetal bovine serum (FBS) or plasma amine oxidase (PAO) induced a sol-gel transition in A9K2 aqueous solutions. The transformation of A9K2 molecules catalyzed by lysyl oxidase (LO) in FBS or PAO accounted for the hydrogelation. Importantly, the enzymatic A9K2 hydrogel displayed high antibacterial ability against both Gram-negative and Gram-positive bacterial strains while showing extremely low mammalian cell cytotoxicity, thus demonstrating good biocompatibility. Under established coculture conditions, the peptide hydrogel showed excellent selectivity by favoring the adherence and spreading of mammalian cells, while killing pathogenic bacteria, thus avoiding bacterial contamination. These advantages endow the enzymatic A9K2 hydrogel with great potential for biomedical applications.

  18. In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

    PubMed

    Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

    2016-01-15

    Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression.

  19. Solid consistency

    NASA Astrophysics Data System (ADS)

    Bordin, Lorenzo; Creminelli, Paolo; Mirbabayi, Mehrdad; Noreña, Jorge

    2017-03-01

    We argue that isotropic scalar fluctuations in solid inflation are adiabatic in the super-horizon limit. During the solid phase this adiabatic mode has peculiar features: constant energy-density slices and comoving slices do not coincide, and their curvatures, parameterized respectively by ζ and Script R, both evolve in time. The existence of this adiabatic mode implies that Maldacena's squeezed limit consistency relation holds after angular average over the long mode. The correlation functions of a long-wavelength spherical scalar mode with several short scalar or tensor modes is fixed by the scaling behavior of the correlators of short modes, independently of the solid inflation action or dynamics of reheating.

  20. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  1. The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity.

    PubMed

    Checovich, Mariana L; Galatro, Andrea; Moriconi, Jorge I; Simontacchi, Marcela; Dubcovsky, Jorge; Santa-María, Guillermo E

    2016-07-01

    TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity.

  2. Crystal structure of β1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.

    PubMed

    Godoy, Andre Schutzer; Camilo, Cesar Moises; Kadowaki, Marco Antonio; Muniz, Heloisa Dos S; Espirito Santo, Melissa; Murakami, Mario Tyago; Nascimento, Alessandro S; Polikarpov, Igor

    2016-11-01

    In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable.

  3. A study on the stability and enzymatic activity of yeast alcohol dehydrogenase in presence of the self-assembling block copolymer Poloxamer 407.

    PubMed

    Pucciarelli, Stefania; Bonacucina, Giulia; Bernabucci, Franco; Cespi, Marco; Mencarelli, Giovanna; De Fronzo, Giusi Serena; Natalini, Paolo; Palmieri, Giovanni Filippo

    2012-05-01

    Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

  4. Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

    PubMed

    Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

    2010-12-01

    l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

  5. Evaluation of Biological and Enzymatic Activity of Soil in a Tropical Dry Forest: Desierto de la Tatacoa (Colombia) with Potential in Mars Terraforming and Other Similar Planets

    NASA Astrophysics Data System (ADS)

    Moreno Moreno, A. N.

    2009-12-01

    Desierto de la Tatacoa has been determined to be a tropical dry forest bioma, which is located at 3° 13" N 75° 13" W. It has a hot thermal floor with 440 msnm of altitude; it has a daily average of 28° C, and a maximum of 40° C, Its annual rainfall total can be upwards of 1250 mm. Its solar sheen has a daily average of 5.8 hours and its relative humidity is between 60% and 65%. Therefore, the life forms presents are very scant, and in certain places, almost void. It was realized a completely random sampling of soil from its surface down to 6 inches deep, of zones without vegetation and with soils highly loaded by oxides of iron in order to determine the number of microorganisms per gram and its subsequent identification. It was measured the soil basal respiration. Besides, it was determined enzymatic activity (catalase, dehydrogenase, phosphatase and urease). Starting with the obtained results, it is developes an alternative towards the study of soil genesis in Mars in particular, and recommendations for same process in other planets. Although the information found in the experiments already realized in Martian soil they demonstrate that doesnt exist any enzymatic activity, the knowledge of the same topic in the soil is proposed as an alternative to problems like carbonic fixing of the dense Martian atmosphere of CO2, the degradation of inorganic compounds amongst other in order to prepare the substratum for later colonization by some life form.

  6. Intracellular amyloid beta interacts with SOD1 and impairs the enzymatic activity of SOD1: implications for the pathogenesis of amyotrophic lateral sclerosis.

    PubMed

    Yoon, Eun Jin; Park, Hyo Jin; Kim, Goo Young; Cho, Hyung Min; Choi, Jung Ha; Park, Hye Yoon; Jang, Ja Young; Rhim, Hyang Shuk; Kang, Seong Man

    2009-09-30

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1), including G93A, were reportedly linked to familial ALS. SOD1 is a key antioxidant enzyme, and is also one of the major targets for oxidative damage in the brains of patients suffering from Alzheimers disease (AD). Several lines of evidence suggest that intracellular amyloid beta (Abeta) is associated with the pathogenesis of AD. In this report we demonstrate that intracellular Abeta directly interacts with SOD1, and that this interaction decreases the enzymatic activity of the enzyme. We observed Abeta-SOD1 aggregates in the perinuclear region of H4 cells, and mapped the SOD1 binding region to Abeta amino acids 26-42. Interestingly, intracellular Ab binds to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. This resulted in considerably less mutant enzymatic activity. Our study implicates a potential role for Abeta in the development of ALS by interacting with the SOD1 G93A mutant.

  7. Influence of crop rotation, intermediate crops, and organic fertilizers on the soil enzymatic activity and humus content in organic farming systems

    NASA Astrophysics Data System (ADS)

    Marcinkeviciene, A.; Boguzas, V.; Balnyte, S.; Pupaliene, R.; Velicka, R.

    2013-02-01

    The influence of crop rotation systems with different portions of nitrogen-fixing crops, intermediate crops, and organic fertilizers on the enzymatic activity and humus content of soils in organic farming was studied. The highest activity of the urease and invertase enzymes was determined in the soil under the crop rotation with 43% nitrogen-fixing crops and with perennial grasses applied twice per rotation. The application of manure and the growing of intermediate crops for green fertilizers did not provide any significant increase in the content of humus. The activity of urease slightly correlated with the humus content ( r = 0.30 at the significance level of 0.05 and r = 0.39 at the significance level of 0.01).

  8. Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells.

    PubMed

    Godoy-Gallardo, Maria; Labay, Cédric; Trikalitis, Vasileios D; Kempen, Paul J; Larsen, Jannik B; Andresen, Thomas L; Hosta-Rigau, Leticia

    2017-02-13

    Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a subcompartmentalized and intracellularly active carrier, a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.

  9. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  10. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    PubMed

    Li, Yan; Burclaff, Joseph; Anderson, James T

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  11. Enzymatic properties and anticancer activity of L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai BKMF-4268D.

    PubMed

    Pokrovsky, Vadim S; Treshalina, Helen M; Lukasheva, Elena V; Sedakova, Ludmila A; Medentzev, Alexander G; Arinbasarova, Anna Yu; Berezov, Temirbolat T

    2013-09-01

    L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (Km for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115-116 kDa; the active dimer consists of two identical 57-58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC50) ranging from 3.0×10 to 7.8×10 U/ml (3.2×10 to 8.2×10 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.

  12. The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

    SciTech Connect

    Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian; Dunn, Christopher A.; Schoeffield, Andrew J.; Bessman, Maurice J.; Amzel, L. Mario

    2011-09-06

    A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.

  13. Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.

    PubMed Central

    Guttridge, K L; Smith, L D; Miledi, R

    1995-01-01

    We cloned the Xenopus laevis form of Gq alpha subunit to study its effects on oocyte maturation. Injection of Xenopus Gq alpha mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (T(in)), due to the opening of Ca(2+)-dependent Cl- channels. The T(in) amplitude developed over several hours and disappeared by 24 hr. Diacylglycerol levels were found to parallel the appearance and disappearance of the T(in). The concurrent decline of T(in) values and diacylglycerol was not due to a failure in the synthesis of Gq alpha protein, which was produced continuously for > 24 hr. After Xenopus Gq alpha mRNA injection, germinal vesicle breakdown (GVBD) was variable (0-100%) in stage 6 oocytes, whereas none of the stage 4 oocytes underwent GVBD. In contrast, stage 6 oocytes injected with mRNA encoding the Go alpha G protein consistently underwent GVBD but did not acquire T(in). Our results show that activation of phospholipase C is not an absolute requisite for the induction of maturation, although in oocytes of some frogs phospholipase C activation can trigger a pathway to GVBD. Images Fig. 7 PMID:7877971

  14. Enzymatic activity of coenzyme B(12) derivatives with altered axial nucleotides: probing the mechanochemical triggering hypothesis in ribonucleotide reductase.

    PubMed

    Brown, K L; Zou, X; Li, J; Chen, G

    2001-11-05

    Theoretical studies (J. Inorg. Biochem. 2001, 83, 121) of the involvement of the bulky 5,6-dimethylbenzimidazole (Dmbz) ligand of coenzyme B(12) (5'-deoxyadenosylcobalamin, AdoCbl) in the mechanism of activation of the carbon-cobalt bond of the coenzyme for homolytic cleavage by AdoCbl-dependent enzymes (the "mechanochemical triggering" mechanisms) have shown that a purely steric, ground-state mechanism can supply only a few kilocalories per mole (of the observed 13-16 kcal mol(-1)) of activation, but that an electronic mechanism, operating to stabilize the transition state, can explain all of the observed catalytic effect. To address these mechanisms experimentally, analogues of AdoCbl in which the Dmbz ligand is replaced by benzimidazole (Ado(Bzim)Cbl) or by imidazole (Ado(Im)Cbl) have been prepared and characterized. Both of these analogues support turnover in the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii at 100% of the activity of AdoCbl itself, but the Ado(Im)Cbl analogue has a significantly higher K(m). 5'-Deoxyadenosylcobinamide, the analogue in which the axial nucleotide has been chemically removed, in contrast, is inactive in the spectrophotometric assay, which indicates that it has at most 1% of the activity of AdoCbl. Stopped-flow spectrophotometric measurements of the formation of cob(II)alamin at the enzyme active site show that RTPR binds Ado(Bzim)Cbl slightly more weakly than it does AdoCbl, but binds Ado(Im)Cbl 8-fold more weakly. While the equilibrium constant for cob(II)alamin formation is nearly the same for Ado(Bzim)Cbl and AdoCbl, it is 5-fold smaller for Ado(Im)Cbl. Finally, the forward rate constant for enzyme-induced Co-C bond homolysis was about the same for Ado(Bzim)Cbl and for AdoCbl but was 17-fold smaller for Ado(Im)Cbl. These results are consistent with a small contribution from ground-state mechanochemical triggering, but they do not in themselves rule out transition-state mechanical

  15. Global histone deacetylase enzymatic activity is an independent prognostic marker associated with a shorter overall survival in chronic lymphocytic leukemia patients.

    PubMed

    Van Damme, Michaël; Crompot, Emerence; Meuleman, Nathalie; Mineur, Philippe; Dessars, Barbara; El Housni, Hakim; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

    2014-10-01

    Histone deacetylases (HDAC) play a crucial role in transcriptional regulation and are often deregulated in many cancers. However, global HDAC enzymatic activity has never been investigated in Chronic Lymphocytic Leukemia (CLL). We measured HDAC activity in protein extracts from CD19+ B-cells purified from 114 CLL patients with a median follow-up of 91 months (range: 11-376). HDAC activity was equivalent in CLL and normal B-cells but higher in patients who died during the study than in living patients (152.1 vs. 65.04 pmol; P = 0.0060). Furthermore, HDAC activity correlated with treatment-free survival (TFS; P = 0.0156) and overall survival (OS; P < 0.0001): patients with low HDAC activity (n = 75) had a median TFS and OS of 101 and > 376 months, respectively, whereas patients with high HDAC activity (n = 39) had a median TFS and OS of 47 and 137 months, respectively. Multivariate analyses indicated that HDAC activity is an independent predictor of OS (hazard ratio = 7.68; P = 0.0017). Finally, HDAC activity increased after B-cell receptor stimulation using IgM, suggesting a role for microenvironment stimuli (n = 10; P = 0.0371). In conclusion, high HDAC activity in CLL B-cells is associated with shorter TFS and OS and is an independent marker of OS, refining the use of other prognostic factors. This work provides a biological base for the use of HDAC inhibitors in CLL treatment.

  16. Communication: Smoothing out excited-state dynamics: Analytical gradients for dynamically weighted complete active space self-consistent field

    SciTech Connect

    Glover, W. J.

    2014-11-07

    State averaged complete active space self-consistent field (SA-CASSCF) is a workhorse for determining the excited-state electronic structure of molecules, particularly for states with multireference character; however, the method suffers from known issues that have prevented its wider adoption. One issue is the presence of discontinuities in potential energy surfaces when a state that is not included in the state averaging crosses with one that is. In this communication I introduce a new dynamical weight with spline (DWS) scheme that mimics SA-CASSCF while removing energy discontinuities due to unweighted state crossings. In addition, analytical gradients for DWS-CASSCF (and other dynamically weighted schemes) are derived for the first time, enabling energy-conserving excited-state ab initio molecular dynamics in instances where SA-CASSCF fails.

  17. The impact of land use intensity and associated pesticide applications on fitness and enzymatic activity in reptiles-A field study.

    PubMed

    Mingo, Valentin; Lötters, Stefan; Wagner, Norman

    2017-03-01

    Environmental pollution and habitat loss are described as underlying causes for population declines in reptiles and especially affect species in agricultural landscapes. Studies dealing with effects of pesticide exposure on reptiles are limited, mainly addressing the orders Testudines and Crocodylia, but largely neglecting the most diverse reptile order Squamata (lizards and snakes). As a consequence, information regarding effects on their organisms, as well as exposure probability and pesticide uptake in the Reptilia h