Sample records for key enzyme responsible

  1. Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

    PubMed

    Pavelka, S

    2014-01-01

    We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

  2. The use of mode of action information in risk assessment: quantitative key events/dose-response framework for modeling the dose-response for key events.

    PubMed

    Simon, Ted W; Simons, S Stoney; Preston, R Julian; Boobis, Alan R; Cohen, Samuel M; Doerrer, Nancy G; Fenner-Crisp, Penelope A; McMullin, Tami S; McQueen, Charlene A; Rowlands, J Craig

    2014-08-01

    The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

  3. Enzyme Sequestration as a Tuning Point in Controlling Response Dynamics of Signalling Networks

    PubMed Central

    Ollivier, Julien F.; Soyer, Orkun S.

    2016-01-01

    Signalling networks result from combinatorial interactions among many enzymes and scaffolding proteins. These complex systems generate response dynamics that are often essential for correct decision-making in cells. Uncovering biochemical design principles that underpin such response dynamics is a prerequisite to understand evolved signalling networks and to design synthetic ones. Here, we use in silico evolution to explore the possible biochemical design space for signalling networks displaying ultrasensitive and adaptive response dynamics. By running evolutionary simulations mimicking different biochemical scenarios, we find that enzyme sequestration emerges as a key mechanism for enabling such dynamics. Inspired by these findings, and to test the role of sequestration, we design a generic, minimalist model of a signalling cycle, featuring two enzymes and a single scaffolding protein. We show that this simple system is capable of displaying both ultrasensitive and adaptive response dynamics. Furthermore, we find that tuning the concentration or kinetics of the sequestering protein can shift system dynamics between these two response types. These empirical results suggest that enzyme sequestration through scaffolding proteins is exploited by evolution to generate diverse response dynamics in signalling networks and could provide an engineering point in synthetic biology applications. PMID:27163612

  4. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

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

  5. Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

    PubMed

    Liu, Lin; Yang, DongFeng; Liang, TongYao; Zhang, HaiHua; He, ZhiGui; Liang, ZongSuo

    2016-09-01

    Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

  6. Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji'an, China.

    PubMed

    Yin, Juxin; Zhang, Daihui; Zhuang, Jianjian; Huang, Yi; Mu, Ying; Lv, Shaowu

    2017-12-11

    Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.

  7. Biosynthesis of abscisic acid in fungi: Identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

    PubMed

    Izquierdo-Bueno, Inmaculada; González-Rodríguez, Victoria E; Simon, Adeline; Dalmais, Bérengère; Pradier, Jean-Marc; Le Pêcheur, Pascal; Mercier, Alex; Walker, Anne-Sophie; Garrido, Carlos; Collado, Isidro González; Viaud, Muriel

    2018-04-30

    While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-α-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes i.e. Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-α-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Enzyme-Responsive Nanomaterials for Controlled Drug Delivery

    PubMed Central

    Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

    2015-01-01

    Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area. PMID:25251024

  9. Enzyme-responsive nanomaterials for controlled drug delivery

    NASA Astrophysics Data System (ADS)

    Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

    2014-10-01

    Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials used for controlled drug release have achieved significant development and have been studied as an important class of drug delivery strategies in nanomedicine. In this review, we describe enzymes such as proteases, phospholipases and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area.

  10. Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

    PubMed

    Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  12. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  13. Regulation-Structured Dynamic Metabolic Model Provides a Potential Mechanism for Delayed Enzyme Response in Denitrification Process

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Song, Hyun-Seob; Thomas, Dennis G.; Stegen, James C.

    In a recent study of denitrification dynamics in hyporheic zone sediments, we observed a significant time lag (up to several days) in enzymatic response to the changes in substrate concentration. To explore an underlying mechanism and understand the interactive dynamics between enzymes and nutrients, we developed a trait-based model that associates a community’s traits with functional enzymes, instead of typically used species guilds (or functional guilds). This enzyme-based formulation allows to collectively describe biogeochemical functions of microbial communities without directly parameterizing the dynamics of species guilds, therefore being scalable to complex communities. As a key component of modeling, we accountedmore » for microbial regulation occurring through transcriptional and translational processes, the dynamics of which was parameterized based on the temporal profiles of enzyme concentrations measured using a new signature peptide-based method. The simulation results using the resulting model showed several days of a time lag in enzymatic responses as observed in experiments. Further, the model showed that the delayed enzymatic reactions could be primarily controlled by transcriptional responses and that the dynamics of transcripts and enzymes are closely correlated. The developed model can serve as a useful tool for predicting biogeochemical processes in natural environments, either independently or through integration with hydrologic flow simulators.« less

  14. Ubiquitin enzymes in the regulation of immune responses.

    PubMed

    Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

    2017-08-01

    Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

  15. Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb, a Key Enzyme in Amylopectin Biosynthesis

    PubMed Central

    Makhmoudova, Amina; Williams, Declan; Brewer, Dyanne; Massey, Sarah; Patterson, Jenelle; Silva, Anjali; Vassall, Kenrick A.; Liu, Fushan; Subedi, Sanjeena; Harauz, George; Siu, K. W. Michael; Tetlow, Ian J.; Emes, Michael J.

    2014-01-01

    Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser649, Ser286, and Ser297. Two Ca2+-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser649 and Ser286 phosphorylation, and K2, responsible for Ser649 and Ser297 phosphorylation. The Ser286 and Ser297 phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser297 forms a stable salt bridge with Arg665, part of a conserved Cys-containing domain in plant branching enzymes. Ser649 conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application. PMID:24550386

  16. Ubiquitin enzymes in the regulation of immune responses

    PubMed Central

    Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

    2017-01-01

    Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

  17. Using soil enzymes to explain observed differences in the response of soil decomposition to nitrogen fertilization

    NASA Astrophysics Data System (ADS)

    Stone, M.; Weiss, M.; Goodale, C. L.

    2010-12-01

    Soil microbes produce extracellular enzymes that degrade a variety of carbon-rich polymers contained within soil organic matter (SOM). These enzymes are key regulators of the terrestrial carbon cycle. However, basic information about the kinetics of extracellular enzymes and key environmental variables that regulate their catalytic ability is lacking. This study aims to clarify the mechanisms by which microbial carbon-degrading enzymes drive different responses to nitrogen (N) fertilization in soil decomposition at two sites with long-term N fertilization experiments, the Bear Brook (BB) forest in Maine and Fernow Forest (FF) in West Virginia. We examined a suite of cellulolytic and lignolytic enzymes that break down common SOM constituents. We hypothesized that enzymes derived from the site with a higher mean annual temperature (FF) would be more heat-tolerant, and retain their catalytic efficiency (Km) as temperature rises, relative to enzymes from the colder environment (BB). We further hypothesized that cellulolytic enzyme activity would be unaffected by N, while oxidative enzyme activity would be suppressed in N-fertilized soils. To test these hypotheses and examine the interactive effects of temperature and N, we measured enzyme activity in unfertilized and N-fertilized soils under a range of laboratory temperature manipulations. Preliminary results show a significant decrease in cellulolytic enzyme efficiency with temperature at the colder site (BB), as well as a significant increase in efficiency due to N-fertilization for two cellulolytic enzymes. Oxidative enzyme activity shows a marginally significant reduction due to N-fertilization at BB. These results suggest that soil warming may produce a negative feedback on carbon turnover in certain climates, while N-fertilization may alter the relative decomposition rates of different soil organic matter constituents. FF activity will be analyzed in a similar manner and the two sites will be compared in order to

  18. Rhodanese Functions as Sulfur Supplier for Key Enzymes in Sulfur Energy Metabolism

    PubMed Central

    Aussignargues, Clément; Giuliani, Marie-Cécile; Infossi, Pascale; Lojou, Elisabeth; Guiral, Marianne; Giudici-Orticoni, Marie-Thérèse; Ilbert, Marianne

    2012-01-01

    How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus. PMID:22496367

  19. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  20. Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen.

    PubMed

    Fuerst, E Patrick; James, Matthew S; Pollard, Anne T; Okubara, Patricia A

    2017-01-01

    Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a .1 was relatively rapid in wild oat ( Avena fatua L.) isoline "M73," with >50% decay after 8 days with almost no decay in wheat ( Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a .1 for 2-3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a .1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a .1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a .1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to

  1. Survey of enzyme activity responsible for phenolic off-flavour production by Dekkera and Brettanomyces yeast.

    PubMed

    Harris, Victoria; Ford, Christopher M; Jiranek, Vladimir; Grbin, Paul R

    2009-01-01

    Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.

  2. 49 CFR 1.13 - OST Key Responsibilities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false OST Key Responsibilities. 1.13 Section 1.13... of the Secretary § 1.13 OST Key Responsibilities. (a) The OST is responsible for: (1) Providing... Government; (5) Encouraging maximum private development of transportation services; (6) Providing responsive...

  3. 49 CFR 1.13 - OST Key Responsibilities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false OST Key Responsibilities. 1.13 Section 1.13... of the Secretary § 1.13 OST Key Responsibilities. (a) The OST is responsible for: (1) Providing... Government; (5) Encouraging maximum private development of transportation services; (6) Providing responsive...

  4. 49 CFR 1.13 - OST Key Responsibilities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false OST Key Responsibilities. 1.13 Section 1.13... of the Secretary § 1.13 OST Key Responsibilities. (a) The OST is responsible for: (1) Providing... Government; (5) Encouraging maximum private development of transportation services; (6) Providing responsive...

  5. Key Building Blocks via Enzyme-Mediated Synthesis

    NASA Astrophysics Data System (ADS)

    Fischer, Thomas; Pietruszka, Jörg

    Biocatalytic approaches to valuable building blocks in organic synthesis have emerged as an important tool in the last few years. While first applications were mainly based on hydrolases, other enzyme classes such as oxidoreductases or lyases moved into the focus of research. Nowadays, a vast number of biotransformations can be found in the chemical and pharmaceutical industries delivering fine chemicals or drugs. The mild reaction conditions, high stereo-, regio-, and chemoselectivities, and the often shortened reaction pathways lead to economical and ecological advantages of enzymatic conversions. Due to the enormous number of enzyme-mediated syntheses, the present chapter is not meant to be a complete review, but to deliver comprehensive insights into well established enzymatic systems and recent advances in the application of enzymes in natural product synthesis. Furthermore, it is focused on the most frequently used enzymes or enzyme classes not covered elsewhere in the present volume.

  6. Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen

    PubMed Central

    Fuerst, E. Patrick; James, Matthew S.; Pollard, Anne T.; Okubara, Patricia A.

    2018-01-01

    Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline “M73,” with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2–3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to

  7. Enzyme action in the regulation of plant hormone responses.

    PubMed

    Westfall, Corey S; Muehler, Ashley M; Jez, Joseph M

    2013-07-05

    Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here, we review new insights into the role of enzyme families, including the SABATH methyltransferases, the methylesterases, the GH3 acyl acid-amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation.

  8. In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

    PubMed

    Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

  9. Enzyme-Responsive Liposomes for the Delivery of Anticancer Drugs

    PubMed Central

    Fouladi, Farnaz; Steffen, Kristine J.; Mallik, Sanku

    2017-01-01

    Liposomes are nanocarriers that deliver the payloads at the target site, leading to therapeutic drug concentrations at the diseased site and reduced toxic effects in healthy tissues. Several approaches have been used to enhance the ability of the nanocarrier to target the specific tissues, including ligand-targeted liposomes and stimuli-responsive liposomes. Ligand-targeted liposomes exhibit higher uptake by the target tissue due to the targeting ligand attached to the surface, while, the stimuli-responsive liposomes do not release their cargo unless they expose to an endogenous or exogenous stimulant at the target site. In this review, we mainly focus on the liposomes that are responsive to pathologically increased levels of enzymes at the target site. Enzyme-responsive liposomes release their cargo upon contact with the enzyme through several destabilization mechanisms: a) structural perturbation in the lipid bilayer, b) removal of a shielding polymer from the surface and increased cellular uptake, c) cleavage of a lipopeptide or lipopolymer incorporated in the bilayer, and d) activation of a prodrug in the liposomes. PMID:28201868

  10. Enzyme-Responsive Liposomes for the Delivery of Anticancer Drugs.

    PubMed

    Fouladi, Farnaz; Steffen, Kristine J; Mallik, Sanku

    2017-04-19

    Liposomes are nanocarriers that deliver the payloads at the target site, leading to therapeutic drug concentrations at the diseased site and reduced toxic effects in healthy tissues. Several approaches have been used to enhance the ability of the nanocarrier to target the specific tissues, including ligand-targeted liposomes and stimuli-responsive liposomes. Ligand-targeted liposomes exhibit higher uptake by the target tissue due to the targeting ligand attached to the surface, while the stimuli-responsive liposomes do not release their cargo unless they expose to an endogenous or exogenous stimulant at the target site. In this review, we mainly focus on the liposomes that are responsive to pathologically increased levels of enzymes at the target site. Enzyme-responsive liposomes release their cargo upon contact with the enzyme through several destabilization mechanisms: (1) structural perturbation in the lipid bilayer, (2) removal of a shielding polymer from the surface and increased cellular uptake, (3) cleavage of a lipopeptide or lipopolymer incorporated in the bilayer, and (4) activation of a prodrug in the liposomes.

  11. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  12. Wild-type and mutated IDH1/2 enzymes and therapy responses.

    PubMed

    Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

    2018-04-01

    Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

  13. Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors

    PubMed Central

    Gabris, Christina; Bengelsdorf, Frank R; Dürre, Peter

    2015-01-01

    This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23–0.99 U mg−1 protein), butyrate kinase (Buk, < 0.03 U mg−1 protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24–7.64 U mg−1 protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH3 and NH4+-N), and a negative dependency can be postulated. Thus, high concentrations of NH3 and NH4+-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities. PMID:26086956

  14. Enzyme-Responsive Delivery of Multiple Proteins with Spatiotemporal Control.

    PubMed

    Zhu, Suwei; Nih, Lina; Carmichael, S Thomas; Lu, Yunfeng; Segura, Tatiana

    2015-06-24

    Orchestrated biological materials such as enzymes and growth factors regulate the growth of tissues and organs. A chirality-controlled, single-protein technology is devised to tailor the spatiotemporally defined delivery of therapeutic proteins in response to natural enzymes present at wound sites. Sustained delivery of one protein and sequential delivery of two proteins are demonstrated for stroke and skin wound healing. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

    PubMed Central

    Solomon, Ariel; Akabayov, Barak; Frenkel, Anatoly; Milla, Marcos E.; Sagi, Irit

    2007-01-01

    Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal–protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design. PMID:17360351

  16. Homogalacturonan-modifying enzymes: structure, expression, and roles in plants

    PubMed Central

    Sénéchal, Fabien; Wattier, Christopher; Rustérucci, Christine; Pelloux, Jérôme

    2014-01-01

    Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses. PMID:25056773

  17. Autoshaping and automaintenance of a key-press response in squirrel monkeys.

    PubMed

    Gamzu, E; Schwam, E

    1974-03-01

    Following exposure for a minimum of 500 to 600 trials, three of four naive squirrel monkeys eventually pressed a response key, illumination of which always preceded delivery of a food pellet. Three other naive monkeys did not press the key when the pellets were delivered randomly with respect to key illumination. Despite some similarities to autoshaping using pigeons, the data indicate many points of difference when squirrel monkeys are used as subjects. Although key-food pairings were shown to be important in the acquisition of the key-press response, they were ineffective in maintaining the response when either a negative response-reinforcer dependency was introduced, or when there was no scheduled response-reinforcer dependency (fixed trial). Not all demonstrations of autoshaping can be considered to be under the control of those processes that are primarily responsible for the phenomena obtained in pigeons.

  18. Autoshaping and automaintenance of a key-press response in squirrel monkeys

    PubMed Central

    Gamzu, Elkan; Schwam, Elias

    1974-01-01

    Following exposure for a minimum of 500 to 600 trials, three of four naive squirrel monkeys eventually pressed a response key, illumination of which always preceded delivery of a food pellet. Three other naive monkeys did not press the key when the pellets were delivered randomly with respect to key illumination. Despite some similarities to autoshaping using pigeons, the data indicate many points of difference when squirrel monkeys are used as subjects. Although key-food pairings were shown to be important in the acquisition of the key-press response, they were ineffective in maintaining the response when either a negative response-reinforcer dependency was introduced, or when there was no scheduled response-reinforcer dependency (fixed trial). Not all demonstrations of autoshaping can be considered to be under the control of those processes that are primarily responsible for the phenomena obtained in pigeons. PMID:16811749

  19. Two-key concurrent responding: response-reinforcement dependencies and blackouts1

    PubMed Central

    Herbert, Emily W.

    1970-01-01

    Two-key concurrent responding was maintained for three pigeons by a single variable-interval 1-minute schedule of reinforcement in conjunction with a random number generator that assigned feeder operations between keys with equal probability. The duration of blackouts was varied between keys when each response initiated a blackout, and grain arranged by the variable-interval schedule was automatically presented after a blackout (Exp. I). In Exp. II every key peck, except for those that produced grain, initiated a blackout, and grain was dependent upon a response following a blackout. For each pigeon in Exp. I and for one pigeon in Exp. II, the relative frequency of responding on a key approximated, i.e., matched, the relative reciprocal of the duration of the blackout interval on that key. In a third experiment, blackouts scheduled on a variable-interval were of equal duration on the two keys. For one key, grain automatically followed each blackout; for the other key, grain was dependent upon a response and never followed a blackout. The relative frequency of responding on the former key, i.e., the delay key, better approximated the negative exponential function obtained by Chung (1965) than the matching function predicted by Chung and Herrnstein (1967). PMID:16811458

  20. Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro.

    PubMed

    Ademiluyi, Adedayo O; Oboh, Ganiyu

    2013-03-01

    This study sought to assess the inhibitory activities of phenolic-rich extracts from soybean on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE) activities in vitro. The free phenolic extract of the soybean was obtained by extraction with 80% acetone, while that of the bound phenolic extract was done by extracting the alkaline and acid hydrolyzed residue with ethyl acetate. The inhibitory action of these extracts on the enzymes activity as well as their antioxidant properties was assessed. Both phenolic-rich extracts inhibited α-amylase, α-glucosidase and ACE enzyme activities in a dose dependent pattern. However, the bound phenolic extract exhibited significantly (P < 0.05) higher α-amylase and ACE inhibition while the free phenolic extract had significantly (P < 0.05) higher α-glucosidase inhibitory activity. Nevertheless, the free phenolic extract had higher α-glucosidase inhibitory activity when compared to that of α-amylase; this property confer an advantage on soybean phenolic-rich extracts over commercial antidiabetic drugs with little or no side effect. And inhibition of ACE suggests the antihypertension potential of soybean phenolic-rich extracts. Furthermore, the enzyme inhibitory activities of the phenolic-rich extracts were not associated with their phenolic content. Therefore, phenolic-rich extracts of soybean could inhibit key-enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (ACE) and thus could explain in part the mechanism by which soybean renders these health promoting effect. Copyright © 2011 Elsevier GmbH. All rights reserved.

  1. Mechanisms, biology and inhibitors of deubiquitinating enzymes.

    PubMed

    Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L

    2007-11-01

    The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

  2. NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying.

    PubMed

    Li, Chun; Sun, Jinwei; Qi, Xiaoxi; Liu, Libo

    2015-01-01

    The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.

  3. Glucose-responsive microgels integrated with enzyme nanocapsules for closed-loop insulin delivery.

    PubMed

    Gu, Zhen; Dang, Tram T; Ma, Minglin; Tang, Benjamin C; Cheng, Hao; Jiang, Shan; Dong, Yizhou; Zhang, Yunlong; Anderson, Daniel G

    2013-08-27

    A glucose-responsive closed-loop insulin delivery system represents the ideal treatment of type 1 diabetes mellitus. In this study, we develop uniform injectable microgels for controlled glucose-responsive release of insulin. Monodisperse microgels (256 ± 18 μm), consisting of a pH-responsive chitosan matrix, enzyme nanocapsules, and recombinant human insulin, were fabricated through a one-step electrospray procedure. Glucose-specific enzymes were covalently encapsulated into the nanocapsules to improve enzymatic stability by protecting from denaturation and immunogenicity as well as to minimize loss due to diffusion from the matrix. The microgel system swelled when subjected to hyperglycemic conditions, as a result of the enzymatic conversion of glucose into gluconic acid and protonation of the chitosan network. Acting as a self-regulating valve system, microgels were adjusted to release insulin at basal release rates under normoglycemic conditions and at higher rates under hyperglycemic conditions. Finally, we demonstrated that these microgels with enzyme nanocapsules facilitate insulin release and result in a reduction of blood glucose levels in a mouse model of type 1 diabetes.

  4. New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria.

    PubMed

    Selmer, Thorsten; Pierik, Antonio J; Heider, Johann

    2005-10-01

    During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

  5. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    NASA Astrophysics Data System (ADS)

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-06-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2‧,3‧,4‧,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2‧,3‧,4‧-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15-45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

  6. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    PubMed Central

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-01-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme. PMID:27263468

  7. Maize Homologs of CCoAOMT and HCT, Two Key Enzymes in Lignin Biosynthesis, Form Complexes with the NLR Rp1 Protein to Modulate the Defense Response1

    PubMed Central

    2016-01-01

    Disease resistance (R) genes encode nucleotide binding Leu-rich-repeat (NLR) proteins that confer resistance to specific pathogens. Upon pathogen recognition they trigger a defense response that usually includes a so-called hypersensitive response (HR), a rapid localized cell death at the site of pathogen infection. Intragenic recombination between two maize (Zea mays) NLRs, Rp1-D and Rp1-dp2, resulted in the formation of a hybrid NLR, Rp1-D21, which confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified genes encoding two key enzymes in lignin biosynthesis, hydroxycinnamoyltransferase (HCT) and caffeoyl CoA O-methyltransferase (CCoAOMT), adjacent to the nucleotide polymorphisms that were highly associated with variation in the severity of Rp1-D21-induced HR. We have previously shown that the two maize HCT homologs suppress the HR conferred by Rp1-D21 in a heterologous system, very likely through physical interaction. Here, we show, similarly, that CCoAOMT2 suppresses the HR induced by either the full-length or by the N-terminal coiled-coil domain of Rp1-D21 also likely via physical interaction and that the metabolic activity of CCoAOMT2 is unlikely to be necessary for its role in suppressing HR. We also demonstrate that CCoAOMT2, HCTs, and Rp1 proteins can form in the same complexes. A model is derived to explain the roles of CCoAOMT and HCT in Rp1-mediated defense resistance. PMID:27208251

  8. Maize Homologs of CCoAOMT and HCT, Two Key Enzymes in Lignin Biosynthesis, Form Complexes with the NLR Rp1 Protein to Modulate the Defense Response.

    PubMed

    Wang, Guan-Feng; Balint-Kurti, Peter J

    2016-07-01

    Disease resistance (R) genes encode nucleotide binding Leu-rich-repeat (NLR) proteins that confer resistance to specific pathogens. Upon pathogen recognition they trigger a defense response that usually includes a so-called hypersensitive response (HR), a rapid localized cell death at the site of pathogen infection. Intragenic recombination between two maize (Zea mays) NLRs, Rp1-D and Rp1-dp2, resulted in the formation of a hybrid NLR, Rp1-D21, which confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified genes encoding two key enzymes in lignin biosynthesis, hydroxycinnamoyltransferase (HCT) and caffeoyl CoA O-methyltransferase (CCoAOMT), adjacent to the nucleotide polymorphisms that were highly associated with variation in the severity of Rp1-D21-induced HR We have previously shown that the two maize HCT homologs suppress the HR conferred by Rp1-D21 in a heterologous system, very likely through physical interaction. Here, we show, similarly, that CCoAOMT2 suppresses the HR induced by either the full-length or by the N-terminal coiled-coil domain of Rp1-D21 also likely via physical interaction and that the metabolic activity of CCoAOMT2 is unlikely to be necessary for its role in suppressing HR. We also demonstrate that CCoAOMT2, HCTs, and Rp1 proteins can form in the same complexes. A model is derived to explain the roles of CCoAOMT and HCT in Rp1-mediated defense resistance. © 2016 American Society of Plant Biologists. All Rights Reserved.

  9. Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

    PubMed Central

    Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

    2013-01-01

    Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

  10. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

    PubMed

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V; Mühlemann, Joëlle K; Bomati, Erin K; Bowman, Marianne E; Dudareva, Natalia; Dixon, Richard A; Noel, Joseph P; Wang, Xiaoqiang

    2014-09-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. © 2014 American Society of Plant Biologists. All rights reserved.

  11. Efficacy of azelaic acid on hepatic key enzymes of carbohydrate metabolism in high fat diet induced type 2 diabetic mice.

    PubMed

    Muthulakshmi, Shanmugam; Saravanan, Ramalingam

    2013-06-01

    Azelaic acid (AzA), a C9 linear α,ω-dicarboxylic acid, is found in whole grains namely wheat, rye, barley, oat seeds and sorghum. The study was performed to investigate whether AzA exerts beneficial effect on hepatic key enzymes of carbohydrate metabolism in high fat diet (HFD) induced type 2 diabetic C57BL/6J mice. C57BL/6J mice were fed high fat diet for 10 weeks and subjected to intragastric administration of various doses (20 mg, 40 mg and 80 mg/kg BW) of AzA daily for the subsequent 5 weeks. Rosiglitazone (RSG) was used as reference drug. Body weight, food intake, plasma glucose, plasma insulin, blood haemoglobin (Hb), blood glycosylated haemoglobin (HbA1c), liver glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes(glucose-6-phosphatase and fructose-1,6-bisphosphatase), liver glycogen, plasma and liver triglycerides were examined in mice fed with normal standard diet (NC), high fat diet (HFD), HFD with AzA (HFD + AzA) and HFD with rosiglitazone (HFD + RSG). Among the three doses, 80 mg/kg BW of AzA was able to positively regulate plasma glucose, insulin, blood HbA1c and haemoglobin levels by significantly increasing the activity of hexokinase and glucose-6-phosphate dehydrogenase and significantly decreasing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase thereby increasing the glycogen content in the liver. From this study, we put forward that AzA could significantly restore the levels of plasma glucose, insulin, HbA1c, Hb, liver glycogen and carbohydrate metabolic key enzymes to near normal in diabetic mice and hence, AzA may be useful as a biomaterial in the development of therapeutic agents against high fat diet induced T2DM. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

    PubMed

    Zou, Huibin; Shi, Mengxun; Zhang, Tongtong; Li, Lei; Li, Liangzhi; Xian, Mo

    2017-10-01

    With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

  13. Geraniol, a natural monoterpene, ameliorates hyperglycemia by attenuating the key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats.

    PubMed

    Babukumar, Sukumar; Vinothkumar, Veerasamy; Sankaranarayanan, Chandrasekaran; Srinivasan, Subramani

    2017-12-01

    Geraniol, an acyclic monoterpene alcohol is found in medicinal plants, is used traditionally for several medical purposes including diabetes. The present study evaluates the antihyperglycemic potential of geraniol on key enzymes of carbohydrate metabolism in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in experimental rats, by a single intraperitoneal (i.p) injection of STZ [40 mg/kg body weight (b.w.)]. Different doses of geraniol (100, 200 and 400 mg/kg b.w.) and glyclazide (5 mg/kg b.w.) were administrated orally to diabetic rats for 45 days. Body weight, food intake, plasma glucose, insulin, blood haemoglobin (Hb), glycosylated haemoglobin (HbA 1c ), hepatic glucose metabolic enzymes and glycogen were examined. The LD 50 value of geraniol is 3600 mg/kg b.w. at oral administration in rats. Administration of geraniol in a dose-dependent manner (100, 200, 400 mg/kg b.w.) and glyclazide (5 mg/kg b.w.) for 45 days significantly improved the levels of insulin, Hb and decreased plasma glucose, HbA 1C in diabetic-treated rats. Geraniol at its effective dose (200 mg/kg b.w.) ameliorated the altered activities of carbohydrate metabolic enzymes near normal effects compared with two other doses (100 and 400 mg/kg b.w.). Geraniol treatment to diabetic rats improved hepatic glycogen content suggesting its anti-hyperglycemic potential. Geraniol supplement was found to preserve the normal histological appearance of hepatic cells and pancreatic β-cells in diabetic rats. The present findings suggest that geraniol can potentially ameliorate key enzymes of glucose metabolism in experimental diabetes even though clinical studies used to evaluate this possibility are warranted.

  14. Key enzymes of the retinoid (visual) cycle in vertebrate retina

    PubMed Central

    Kiser, Philip D.; Golczak, Marcin; Maeda, Akiko; Palczewski, Krzysztof

    2011-01-01

    A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. PMID:21447403

  15. Scaling-up HIV responses with key populations in West Africa.

    PubMed

    Wheeler, Tisha; Wolf, R Cameron; Kapesa, Laurent; Cheng Surdo, Alison; Dallabetta, Gina

    2015-03-01

    Despite decades of HIV responses in pockets of West and Central Africa (WCA), the HIV response with key populations remains an understudied area. Recently, there has been a proliferation of studies highlighting epidemiologic and behavioral data that challenge attitudes of complacency among donors and country governments uncomfortable in addressing key populations. The articles in this series highlight new studies that provide a better understanding of the epidemiologic and structural burden facing key populations in the WCA region and how to improve responses through more effective targeting. Key populations face pervasive structural barriers including institutional and sexual violence and an intersection of stigma, criminalization, and marginalization as sexual minorities. Despite decades of smaller interventions that have shown the importance of integrated services for key populations, there remains incongruent provision of outreach or testing or family planning pointing to sustained risk. There remains an incongruent resource provision for key populations where they shoulder the burden of HIV and their access to services alone could turn around HIV epidemics within the region. These proximal and distal determinants must be addressed in regional efforts, led by the community, and resourced for scale, targeting those most at risk for the acquisition and transmission of HIV. This special issue builds the knowledge base for the region focusing on interventions that remove barriers to service access including treatment uptake for those living with HIV. Better analysis and use of data for strategic planning are shown to lead to more effective targeting of prevention, care, and HIV treatment programs with key populations. These articles further demonstrate the immediate need for comprehensive action to address HIV among key populations throughout the WCA region.

  16. Inhibitory activity on type 2 diabetes and hypertension key-enzymes, and antioxidant capacity of Veronica persica phenolic-rich extracts.

    PubMed

    Sharifi-Rad, M; Tayeboon, G S; Sharifi-Rad, J; Iriti, M; Varoni, E M; Razazi, S

    2016-05-30

    Veronica genus (Plantaginaceae) is broadly distributed in different habitats. In this study, the inhibitory activity of free soluble and conjugated phenolic extracts of Veronica persica on key enzymes associated to type 2 diabetes (α-glucosidase and α-amylase) and hypertension (angiotensin I converting enzyme, ACE) was assessed, as well as their antioxidant power. Our results showed that both the extracts inhibited α-amylase, α-glucosidase and ACE in a dose-dependent manner. In particular, free phenolic extract significantly (P<0.05) inhibited α-glucosidase (IC50 532.97 µg/mL), whereas conjugated phenolic extract significantly (P<0.05) inhibited α-amylase (IC50 489.73 µg/mL) and ACE (290.06 µg/mL). The enzyme inhibitory activities of the extracts were not associated with their phenolic content. Anyway, the inhibition of α-amylase, α-glucosidase and ACE, along with the antioxidant capacity of the phenolic-rich extracts, could represent a putative mechanism through which V. persica exerts its antidiabetes and antihypertension effects.

  17. Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response1

    PubMed Central

    Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A.; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J.

    2015-01-01

    In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. PMID:26373661

  18. Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response.

    PubMed

    Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J

    2015-11-01

    In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. © 2015 American Society of Plant Biologists. All Rights Reserved.

  19. Metabolic enzymes: key modulators of functionality in cancer stem-like cells.

    PubMed

    Dong, Bo-Wen; Qin, Guang-Ming; Luo, Yan; Mao, Jian-Shan

    2017-02-21

    Cancer Stem-like Cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity and are important for the initiation, progression and recurrence of cancer diseases. The metabolic profile of CSCs is consistent with their stem-like properties. Studies have indicated that enzymes, the main regulators of cellular metabolism, dictate functionalities of CSCs in both catalysis-dependent and catalysis-independent manners. This paper reviews diverse studies of metabolic enzymes, and describes the effects of these enzymes on metabolic adaptation, gene transcription and signal transduction, in CSCs.

  20. Metabolic enzymes: key modulators of functionality in cancer stem-like cells

    PubMed Central

    Dong, Bo-Wen; Qin, Guang-Ming; Luo, Yan; Mao, Jian-Shan

    2017-01-01

    Cancer Stem-like Cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity and are important for the initiation, progression and recurrence of cancer diseases. The metabolic profile of CSCs is consistent with their stem-like properties. Studies have indicated that enzymes, the main regulators of cellular metabolism, dictate functionalities of CSCs in both catalysis-dependent and catalysis-independent manners. This paper reviews diverse studies of metabolic enzymes, and describes the effects of these enzymes on metabolic adaptation, gene transcription and signal transduction, in CSCs. PMID:28009990

  1. Keys and seats: Spatial response coding underlying the joint spatial compatibility effect.

    PubMed

    Dittrich, Kerstin; Dolk, Thomas; Rothe-Wulf, Annelie; Klauer, Karl Christoph; Prinz, Wolfgang

    2013-11-01

    Spatial compatibility effects (SCEs) are typically observed when participants have to execute spatially defined responses to nonspatial stimulus features (e.g., the color red or green) that randomly appear to the left and the right. Whereas a spatial correspondence of stimulus and response features facilitates response execution, a noncorrespondence impairs task performance. Interestingly, the SCE is drastically reduced when a single participant responds to one stimulus feature (e.g., green) by operating only one response key (individual go/no-go task), whereas a full-blown SCE is observed when the task is distributed between two participants (joint go/no-go task). This joint SCE (a.k.a. the social Simon effect) has previously been explained by action/task co-representation, whereas alternative accounts ascribe joint SCEs to spatial components inherent in joint go/no-go tasks that allow participants to code their responses spatially. Although increasing evidence supports the idea that spatial rather than social aspects are responsible for joint SCEs emerging, it is still unclear to which component(s) the spatial coding refers to: the spatial orientation of response keys, the spatial orientation of responding agents, or both. By varying the spatial orientation of the responding agents (Exp. 1) and of the response keys (Exp. 2), independent of the spatial orientation of the stimuli, in the present study we found joint SCEs only when both the seating and the response key alignment matched the stimulus alignment. These results provide evidence that spatial response coding refers not only to the response key arrangement, but also to the-often neglected-spatial orientation of the responding agents.

  2. Duodenal-jejunal bypass surgery up-regulates the expression of the hepatic insulin signaling proteins and the key regulatory enzymes of intestinal gluconeogenesis in diabetic Goto-Kakizaki rats.

    PubMed

    Sun, Dong; Wang, Kexin; Yan, Zhibo; Zhang, Guangyong; Liu, Shaozhuang; Liu, Fengjun; Hu, Chunxiao; Hu, Sanyuan

    2013-11-01

    Duodenal-jejunal bypass (DJB), which is not routinely applied in metabolic surgery, is an effective surgical procedure in terms of type 2 diabetes mellitus resolution. However, the underlying mechanisms are still undefined. Our aim was to investigate the diabetic improvement by DJB and to explore the changes in hepatic insulin signaling proteins and regulatory enzymes of gluconeogenesis after DJB in a non-obese diabetic rat model. Sixteen adult male Goto-Kakizaki rats were randomly divided into DJB and sham-operated groups. The body weight, food intake, hormone levels, and glucose metabolism were measured. The levels of protein expression and phosphorylation of insulin receptor-beta (IR-β) and insulin receptor substrate 2 (IRS-2) were evaluated in the liver. We also detected the expression of key regulatory enzymes of gluconeogenesis [phosphoenoylpyruvate carboxykinase-1 (PCK1), glucose-6-phosphatase-alpha (G6Pase-α)] in small intestine and liver. DJB induced significant diabetic improvement with higher postprandial glucagons-like peptide 1, peptide YY, and insulin levels, but without weight loss. The DJB group exhibited increased expression and phosphorylation of IR-β and IRS-2 in liver, up-regulated the expression of PCK1 and G6Pase-α in small intestine, and down-regulated the expression of these enzymes in liver. DJB is effective in up-regulating the expression of the key proteins in the hepatic insulin signaling pathway and the key regulatory enzymes of intestinal gluconeogenesis and down-regulating the expression of the key regulatory enzymes of hepatic gluconeogenesis without weight loss. Our study helps to reveal the potential role of hepatic insulin signaling pathway and intestinal gluconeogenesis in ameliorating insulin resistance after metabolic surgery.

  3. Selective Discrimination of Key Enzymes of Pathogenic and Nonpathogenic Bacteria on Autonomously Reporting Shape-Encoded Hydrogel Patterns.

    PubMed

    Jia, Zhiyuan; Sukker, Issa; Müller, Mareike; Schönherr, Holger

    2018-02-14

    This work reports on a new approach to rapidly and selectively detect and discriminate enzymes of pathogenic from those of nonpathogenic bacteria using a patterned autonomously reporting hydrogel on a transparent support, in which the selectivity has been encoded by the pattern shape to enable facile detection by a color change at one single wavelength. In particular, enzyme-responsive chitosan hydrogel layers that report the presence of the enzymes β-glucuronidase (β-Gus) and β-galactosidase (β-Gal), produced by the nonvirulent Escherichia coli K12 and the food-borne biosafety level 3 pathogen enterohemorrhagic E. coli, respectively, via the blue color of an indigo dye were patterned by two complementary strategies. The comparison of the functionalization of patterned chitosan patches on a solid support with two chromogenic substrates on one hand and the area-selective conjugation of the substrates on the other hand showed that the two characteristic enzymes could indeed be rapidly and selectively discriminated. The limits of detection of the highly stable sensing layers for an observation time of 60 min using a spectrophotometer correspond to enzyme concentrations of β-Gus and β-Gal of ≤5 and ≤3 nM, respectively, and to ≤62 and ≤33 nM for bare eye detection in nonoptimized sensor patches. These results confirm the applicability of this approach, which is compatible with the simple measurement of optical density at one single wavelength only as well as with parallel, multiplexed detection, to differentiate the enzymes secreted by a highly pathogenic E. coli from a nonpathogenic E. coli on the basis of specifically secreted enzymes. Hence, a general approach for the rapid and selective detection of enzymes of different bacterial species for potential applications in food safety as well as point-of-care microbiological diagnostics is described.

  4. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  5. Salt stress induced lipid accumulation in heterotrophic culture cells of Chlorella protothecoides: Mechanisms based on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration.

    PubMed

    Wang, Tao; Ge, Haiyan; Liu, Tingting; Tian, Xiwei; Wang, Zejian; Guo, Meijin; Chu, Ju; Zhuang, Yingping

    2016-06-20

    Salt stress as an effective stress factor that could improve the lipid content and lipid yield of glucose in the heterotrophic culture cells of Chlorella protothecoides was demonstrated in this study. The highest lipid content of 41.2% and lipid yield of 185.8mg/g were obtained when C. protothecoides was stressed under 30g/L NaCl condition at its late logarithmic growth phase. Moreover, the effects of salt and osmotic stress on lipid accumulation were comparatively analyzed, and it was found that the effects of NaCl and KCl stress had no significant differences at the same osmolarity level of 1150mOsm/kg with lipid contents of 41.7 and 40.8% as well as lipid yields of 192.9 and 186.8mg/g, respectively, whereas these results were obviously higher than those obtained under the iso-osmotic glycerol and sorbitol stresses. Furthermore, basing on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration, the superior performance of salt stress driving lipid over-synthesis was probably ascribed to the more ROS production as a result of additional ion effect besides the osmotic effect, subsequently mediating the alteration from carbohydrate storage to lipid accumulation in signal transduction process of C. protothecoides. Copyright © 2016. Published by Elsevier B.V.

  6. [Importance of the 11β-hydroxysteroid dehydrogenase enzyme in clinical disorders].

    PubMed

    Feldman, Karolina; Likó, István; Nagy, Zsolt; Szappanos, Agnes; Grolmusz, Vince Kornél; Tóth, Miklós; Rácz, Károly; Patócs, Attila

    2013-02-24

    Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11β-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11β-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

  7. Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

    PubMed Central

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-01-01

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mm, and a specific activity (Vmax) of 20.0 μmol min−1 mg−1 corresponding to a kcat of 7.7 s−1. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase. PMID:25228698

  8. Exposure to leachate from municipal battery recycling site: implication as key inhibitor of steroidogenic enzymes and risk factor of prostate damage in rats.

    PubMed

    Akintunde, Jacob K; Oboh, G

    2013-01-01

    Few or no studies have measured the effect of short- and long-term exposure to industrial leachate. Mature male Wistar strain albino rats (175-220 g) underwent sub-chronic exposure to leachate from the Elewi Odo municipal battery recycling site (EOMABRL) via oral administration for a period of 60 days at different doses (20%, 40%, 60%, 80%, and 100%) per kilogram of body weight to evaluate the toxic effects of the leachate on male reproductive function using steroidogenic enzymes and biomarkers of prostate damage. Control groups were treated equally but were given distilled water instead of the leachate. After the treatment periods, results showed that the treatment induced systemic toxicity at the doses tested by causing a significant (p<0.05) loss in absolute body weight and decline in growth rate. There was a marked significant decrease (p<0.05) in testicular activities of Δ(5)-3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase. Conversely, the activity of prostatic acid phosphatase, a key marker enzyme for prostrate damage was significantly (p<0.05) elevated in the treated rats. Similarly, the administration of EOMABRL significantly (p<0.05) exacerbated the activity of total acid phosphatase with concomitant increase in the activity of prostatic alkaline phosphatase. These findings conclude that exposure to leachate from a battery recycling site induces sub-chronic testicular toxicity by inhibiting key steroidogenic enzymes and activating key markers linked with prostate damage/cancer in rats.

  9. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

    PubMed Central

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

    2015-01-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

  10. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

    PubMed

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

    2015-12-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  11. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    PubMed

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  12. Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybrids.

    PubMed

    Ikeda, Masato; Tanida, Tatsuya; Yoshii, Tatsuyuki; Kurotani, Kazuya; Onogi, Shoji; Urayama, Kenji; Hamachi, Itaru

    2014-06-01

    Soft materials that exhibit stimuli-responsive behaviour under aqueous conditions (such as supramolecular hydrogels composed of self-assembled nanofibres) have many potential biological applications. However, designing a macroscopic response to structurally complex biochemical stimuli in these materials still remains a challenge. Here we show that redox-responsive peptide-based hydrogels have the ability to encapsulate enzymes and still retain their activities. Moreover, cooperative coupling of enzymatic reactions with the gel response enables us to construct unique stimuli-responsive soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel-sol response (even to biological samples) is visible to the naked eye. Furthermore, we built Boolean logic gates (OR and AND) into the hydrogel-enzyme hybrid materials, which were able to sense simultaneously plural specific biochemicals and execute a controlled drug release in accordance with the logic operation. The intelligent soft materials that we have developed may prove valuable in future medical diagnostics or treatments.

  13. Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybrids

    NASA Astrophysics Data System (ADS)

    Ikeda, Masato; Tanida, Tatsuya; Yoshii, Tatsuyuki; Kurotani, Kazuya; Onogi, Shoji; Urayama, Kenji; Hamachi, Itaru

    2014-06-01

    Soft materials that exhibit stimuli-responsive behaviour under aqueous conditions (such as supramolecular hydrogels composed of self-assembled nanofibres) have many potential biological applications. However, designing a macroscopic response to structurally complex biochemical stimuli in these materials still remains a challenge. Here we show that redox-responsive peptide-based hydrogels have the ability to encapsulate enzymes and still retain their activities. Moreover, cooperative coupling of enzymatic reactions with the gel response enables us to construct unique stimuli-responsive soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel-sol response (even to biological samples) is visible to the naked eye. Furthermore, we built Boolean logic gates (OR and AND) into the hydrogel-enzyme hybrid materials, which were able to sense simultaneously plural specific biochemicals and execute a controlled drug release in accordance with the logic operation. The intelligent soft materials that we have developed may prove valuable in future medical diagnostics or treatments.

  14. Angiotensin converting enzyme over expression in myelocytes enhances the immune response

    PubMed Central

    Bernstein, Kenneth E.; Gonzalez-Villalobos, Romer A.; Giani, Jorge F.; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D.; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z.

    2015-01-01

    Angiotensin converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis and several aspects of the immune response. ACE 10/10 mice over express ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization towards a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with either melanoma, bacterial infection or Alzheimer’s disease. The ACE 10/10 mice suggest that enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges. PMID:24633750

  15. Copper stress and filamentous fungus Humicola lutea 103 - ultrastructural changes and activities of key metabolic enzymes.

    PubMed

    Krumova, Ekaterina Ts; Stoitsova, Stoyanka R; Paunova-Krasteva, Tsvetelina S; Pashova, Svetlana B; Angelova, Maria B

    2012-12-01

    Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL(-1) Cu(2+) under submerged cultivation. To prevent the consequences of copper overload, microorganisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. In spite of this avoidance strategy, high heavy-metal concentrations caused distinct and widespread ultrastructural alterations in H. lutea. The mitochondria were the first and main target of the toxic action. The effect of copper on activities of the key enzymes (hexokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase) included in the 3 main metabolic pathways, glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, was investigated. High metal concentrations exhibited a dramatic negative effect on hexokinase, while the other 3 enzymes showed a significant and dose-dependent stimulation. On the basis of the present and previous results we concluded that the copper-induced oxidative stress plays an important role in the fungal tolerance to high Cu (2+) concentrations.

  16. The roles of ubiquitin modifying enzymes in neoplastic disease.

    PubMed

    Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam

    2017-12-01

    The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. A Key Role for Old Yellow Enzyme in the Metabolism of Drugs by Trypanosoma cruzi

    PubMed Central

    Kubata, Bruno Kilunga; Kabututu, Zakayi; Nozaki, Tomoyoshi; Munday, Craig J.; Fukuzumi, Shunichi; Ohkubo, Kei; Lazarus, Michael; Maruyama, Toshihiko; Martin, Samuel K.; Duszenko, Michael; Urade, Yoshihiro

    2002-01-01

    Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of molecules essential in parasite metabolism of the anti-chagasic drugs are fundamental for the development of new strategies for rational drug design and/or the improvement of the current chemotherapy. While searching for a prostaglandin (PG) F2α synthase homologue, we have identified a novel “old yellow enzyme” from T. cruzi (TcOYE), cloned its cDNA, and overexpressed the recombinant enzyme. Here, we show that TcOYE reduced 9,11-endoperoxide PGH2 to PGF2α as well as a variety of trypanocidal drugs. By electron spin resonance experiments, we found that TcOYE specifically catalyzed one-electron reduction of menadione and β-lapachone to semiquinone-free radicals with concomitant generation of superoxide radical anions, while catalyzing solely the two-electron reduction of nifurtimox and 4-nitroquinoline-N-oxide drugs without free radical production. Interestingly, immunoprecipitation experiments revealed that anti-TcOYE polyclonal antibody abolished major reductase activities of the lysates toward these drugs, identifying TcOYE as a key drug-metabolizing enzyme by which quinone drugs have their mechanism of action. PMID:12417633

  18. Electrocatalytic tuning of biosensing response through electrostatic or hydrophobic enzyme-graphene oxide interactions.

    PubMed

    Baptista-Pires, Luis; Pérez-López, Briza; Mayorga-Martinez, Carmen C; Morales-Narváez, Eden; Domingo, Neus; Esplandiu, Maria Jose; Alzina, Francesc; Sotomayor-Torres, Clivia M; Merkoçi, Arben

    2014-11-15

    The effect of graphene oxidative grades upon the conductivity and hydrophobicity and consequently the influence on an enzymatic biosensing response is presented. The electrochemical responses of reduced graphene oxide (rGO) have been compared with the responses obtained from the oxide form (oGO) and their performances have been accordingly discussed with various evidences obtained by optical techniques. We used tyrosinase enzyme as a proof of concept receptor with interest for phenolic compounds detection through its direct adsorption onto a screen-printed carbon electrode previously modified with oGO or rGO with a carbon-oxygen ratio of 1.07 or 1.53 respectively. Different levels of oGO directly affect the (bio)conjugation properties of the biosensor due to changes at enzyme/graphene oxide interface coming from the various electrostatic or hydrophobic interactions with biomolecules. The developed biosensor was capable of reaching a limit of detection of 0.01 nM catechol. This tuning capability of the biosensor response can be of interest for building several other biosensors, including immunosensors and DNA sensors for various applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis.

    PubMed

    Turchetto-Zolet, Andreia C; Maraschin, Felipe S; de Morais, Guilherme L; Cagliari, Alexandro; Andrade, Cláudia M B; Margis-Pinheiro, Marcia; Margis, Rogerio

    2011-09-20

    Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa. Overall, the data show that

  20. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis

    PubMed Central

    2011-01-01

    Background Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. Results We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. Conclusions In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa

  1. Predicting tumor responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumor homogenates: implications for enzyme-directed bioreductive drug development.

    PubMed

    Phillips, R M; Burger, A M; Loadman, P M; Jarrett, C M; Swaine, D J; Fiebig, H H

    2000-11-15

    Mitomycin C (MMC) is a clinically used anticancer drug that is reduced to cytotoxic metabolites by cellular reductases via a process known as bioreductive drug activation. The identification of key enzymes responsible for drug activation has been investigated extensively with the ultimate aim of tailoring drug administration to patients whose tumors possess the biochemical machinery required for drug activation. In the case of MMC, considerable interest has been centered upon the enzyme DT-diaphorase (DTD) although conflicting reports of good and poor correlations between enzyme activity and response in vitro and in vivo have been published. The principle aim of this study was to provide a definitive answer to the question of whether tumor response to MMC could be predicted on the basis of DTD activity in a large panel of human tumor xenografts. DTD levels were measured in 45 human tumor xenografts that had been characterized previously in terms of their sensitivity to MMC in vitro and in vivo (the in vivo response profile to MMC was taken from work published previously). A poor correlation between DTD activity and antitumor activity in vitro as well as in vivo was obtained. This study also assessed the predictive value of an alternative approach based upon the ability of tumor homogenates to metabolize MMC. This approach is based on the premise that the overall rate of MMC metabolism may provide a better indicator of response than single enzyme measurements. MMC metabolism was evaluated in tumor homogenates (clarified by centrifugation at 1000 x g for 1 min) by measuring the disappearance of the parent compound by HPLC. In responsive [T/C <10% (T/C defined as the relative size of treated and control tumors)] and resistant (T/C >50%) tumors, the mean half life of MMC was 75+/-48.3 and 280+/-129.6 min, respectively. The difference between the two groups was statistically significant (P < 0.005). In conclusion, these results unequivocally demonstrate that response to

  2. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    PubMed

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  3. Fabrication of Propeller-Shaped Supra-amphiphile for Construction of Enzyme-Responsive Fluorescent Vesicles.

    PubMed

    Li, Jie; Liu, Kaerdun; Han, Yuchun; Tang, Ben Zhong; Huang, Jianbin; Yan, Yun

    2016-10-04

    Propeller-shaped molecules have been recognized to display fantastic AIE (aggregation induced emission), but they can hardly self-assemble into nanostructures. Herein, we for the first time report that ionic complexation between a water-soluble tetrapheneyl derivative and an enzyme substrate in aqueous media produces a propeller-shaped supra-amphiphile that self-assembles into enzyme responsive fluorescent vesicles. The supra-amphiphile was fabricated upon complexation between a water-soluble propeller-shaped AIE luminogen TPE-BPA and myristoylcholine chloride (MChCl) in aqueous media. MChCl filled in the intramolecular voids of propeller-shaped TPE-BPA upon supra-amphiphile formation, which endows the supra-amphiphile superior self-assembling ability to the component molecules thus leading to the formation of fluorescent vesicles. Because MChCl is the substrate of cholinesterases, the vesicles dissemble in the presence of cholinesterases, and the fluorescent intensity can be correlated to the level of enzymes. The resulting fluorescent vesicles may be used to recognize the site of Alzheimer's disease, to encapsulate the enzyme inhibitor, and to release the inhibitor at the disease site.

  4. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maltz, Lauren

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

  5. Marine Sponges and Bacteria as Challenging Sources of Enzyme Inhibitors for Pharmacological Applications

    PubMed Central

    Ruocco, Nadia; Costantini, Susan; Palumbo, Flora; Costantini, Maria

    2017-01-01

    Enzymes play key roles in different cellular processes, for example, in signal transduction, cell differentiation and proliferation, metabolic processes, DNA damage repair, apoptosis, and response to stress. A deregulation of enzymes has been considered one of the first causes of several diseases, including cancers. In the last several years, enzyme inhibitors, being good candidates as drugs in the pathogenic processes, have received an increasing amount of attention for their potential application in pharmacology. The marine environment is considered a challenging source of enzyme inhibitors for pharmacological applications. In this review, we report on secondary metabolites with enzyme inhibitory activity, focusing our attention on marine sponges and bacteria as promising sources. In the case of sponges, we only reported the kinase inhibitors, because this class was the most representative isolated so far from these marine organisms. PMID:28604647

  6. Modeling the Response of Soil Organic Matter Decomposition to Warming: Effects of Dynamical Enzyme Productivity and Nuanced Representation of Respiration.

    NASA Astrophysics Data System (ADS)

    Sihi, D.; Gerber, S.; Inglett, K. S.; Inglett, P.

    2014-12-01

    Recent development in modeling soil organic carbon (SOC) decomposition includes the explicit incorporation of enzyme and microbial dynamics. A characteristic of these models is a feedback between substrate and consumers which is absent in traditional first order decay models. Second, microbial decomposition models incorporate carbon use efficiency (CUE) as a function of temperature which proved to be critical to prediction of SOC with warming. Our main goal is to explore microbial decomposition models with respect to responses of microbes to enzyme activity, costs to enzyme production, and to incorporation of growth vs. maintenance respiration. In order to simplify the modeling setup we assumed quick adjustment of enzyme activity and depolymerized carbon to microbial and SOC pools. Enzyme activity plays an important role to decomposition if its production is scaled to microbial biomass. In fact if microbes are allowed to optimize enzyme productivity the microbial enzyme model becomes unstable. Thus if the assumption of enzyme productivity is relaxed, other limiting factors must come into play. To stabilize the model, we account for two feedbacks that include cost of enzyme production and diminishing return of depolymerization with increasing enzyme concentration and activity. These feedback mechanisms caused the model to behave in a similar way to traditional, first order decay models. Most importantly, we found, that under warming, the changes in SOC carbon were more severe in enzyme synthesis is costly. In turn, carbon use efficiency (CUE) and its dynamical response to temperature is mainly determined by 1) the rate of turnover of microbes 2) the partitioning of dead microbial matter into different quality pools, and 3) and whether growth, maintenance respiration and microbial death rate have distinct responses to changes in temperature. Abbreviations: p: decay of enzyme, g: coefficient for growth respiration, : fraction of material from microbial turnover that

  7. Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

    PubMed

    Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

    2014-03-05

    The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Fabry disease, enzyme replacement therapy and the significance of antibody responses.

    PubMed

    Deegan, Patrick B

    2012-03-01

    Fabry disease is an X-linked disorder caused by a deficiency of α-galactosidase A. This leads to a progressive accumulation of globotriaosylceramide in tissues throughout the body. Cardiac, renal and neurological manifestations are common and life expectancy is significantly reduced relative to the general population. Management of Fabry disease involves the administration of intravenous enzyme replacement therapy (ERT). Two forms - agalsidase alfa and agalsidase beta - have been licensed in certain jurisdictions and are generally well tolerated; however, some patients develop antibodies to the infused enzyme, which may impair the efficacy and safety of treatment. Agalsidase alfa and agalsidase beta are produced in different systems; this leads to certain differences in post-translational modification that may affect immunogenicity. Immunoglobulin (Ig) G antibodies have frequently been reported in patients with Fabry disease receiving ERT; IgG responses are reported in a greater proportion of patients receiving agalsidase beta than in patients receiving agalsidase alfa. IgE antibodies are less common than IgG antibodies, and have not been observed in patients receiving agalsidase alfa. However, these data are difficult to interpret due to methodological differences in the assessment of seropositivity, and in the doses of enzyme used. The clinical impact of the development of IgG antibodies to ERT in patients with Fabry disease remains unclear, due to lack of data and to the marked heterogeneity of patients both in terms of disease manifestations and response to therapy. Further studies that examine the development of antibodies in patients with Fabry disease and the potential impact of such antibodies on the outcome of ERT are necessary.

  9. Structural aspects of denitrifying enzymes.

    PubMed

    Moura, I; Moura, J J

    2001-04-01

    The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

  10. Enzyme Informatics

    PubMed Central

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

    2012-01-01

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

  11. Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

    PubMed

    Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

    2017-10-12

    Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

  12. Gallium Nitrate Is Efficacious in Murine Models of Tuberculosis and Inhibits Key Bacterial Fe-Dependent Enzymes

    PubMed Central

    Olakanmi, Oyebode; Kesavalu, Banurekha; Pasula, Rajamouli; Abdalla, Maher Y.; Schlesinger, Larry S.

    2013-01-01

    Acquiring iron (Fe) is critical to the metabolism and growth of Mycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga3+ from Fe3+. Unlike Fe3+, Ga3+ cannot be physiologically reduced to Ga2+. Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth of M. tuberculosis in broth and within cultured human macrophages. We now report that Ga(NO3)3 shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally with M. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3 died. All Ga(NO3)3-treated mice survived. M. tuberculosis CFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3 treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga on M. tuberculosis ribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreased M. tuberculosis RR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3 shows efficacy in murine M. tuberculosis infection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Ga's mechanism of action and to optimize delivery forms for possible therapeutic uses in humans. PMID:24060870

  13. Medium Optimization for the Production of Fibrinolytic Enzyme by Paenibacillus sp. IND8 Using Response Surface Methodology

    PubMed Central

    Prakash Vincent, Samuel Gnana

    2014-01-01

    Production of fibrinolytic enzyme by a newly isolated Paenibacillus sp. IND8 was optimized using wheat bran in solid state fermentation. A 25 full factorial design (first-order model) was applied to elucidate the key factors as moisture, pH, sucrose, yeast extract, and sodium dihydrogen phosphate. Statistical analysis of the results has shown that moisture, sucrose, and sodium dihydrogen phosphate have the most significant effects on fibrinolytic enzymes production (P < 0.05). Central composite design (CCD) was used to determine the optimal concentrations of these three components and the experimental results were fitted with a second-order polynomial model at 95% level (P < 0.05). Overall, 4.5-fold increase in fibrinolytic enzyme production was achieved in the optimized medium as compared with the unoptimized medium. PMID:24523635

  14. Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

    PubMed Central

    Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

  15. The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

    PubMed

    Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

    2011-04-01

    Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Modulatory effects of naringin on hepatic key enzymes of carbohydrate metabolism in high-fat diet/low-dose streptozotocin-induced diabetes in rats.

    PubMed

    Pari, Leelavinothan; Chandramohan, Ramasamy

    2017-07-01

    We evaluated the modulatory effects of naringin on altered hepatic key enzymes of carbohydrate metabolism in high-fat diet/low-dose streptozotocin-induced diabetic rats. Oral treatment of naringin at a doses of 20, 40 and 80 mg/kg body weight to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, blood glycosylated hemoglobin and increase in the levels of plasma insulin and blood hemoglobin. The altered activities of the hepatic key enzymes of carbohydrate metabolism such as hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase, glycogen phosphorylase and glycogen content of diabetic rats were significantly reverted to near normal levels by the treatment of naringin in a dose-dependent manner. Naringin at a dose of 80 mg/kg body weight showed the highest significant effect than the other two doses (20 and 40 mg/kg). Further, immunohistochemical observation of pancreas revealed that naringin-treated diabetic rats showed the increased number of insulin immunoreactive β-cells, which confirmed the biochemical findings. These findings revealed that naringin has potential antihyperglycemic activity in high-fat diet/low-dose streptozotocin-induced diabetic rats.

  17. Allosteric regulation of epigenetic modifying enzymes.

    PubMed

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

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

  18. ChoiceKey: a real-time speech recognition program for psychology experiments with a small response set.

    PubMed

    Donkin, Christopher; Brown, Scott D; Heathcote, Andrew

    2009-02-01

    Psychological experiments often collect choice responses using buttonpresses. However, spoken responses are useful in many cases-for example, when working with special clinical populations, or when a paradigm demands vocalization, or when accurate response time measurements are desired. In these cases, spoken responses are typically collected using a voice key, which usually involves manual coding by experimenters in a tedious and error-prone manner. We describe ChoiceKey, an open-source speech recognition package for MATLAB. It can be optimized by training for small response sets and different speakers. We show ChoiceKey to be reliable with minimal training for most participants in experiments with two different responses. Problems presented by individual differences, and occasional atypical responses, are examined, and extensions to larger response sets are explored. The ChoiceKey source files and instructions may be downloaded as supplemental materials for this article from brm.psychonomic-journals.org/content/supplemental.

  19. Phase I to II cross-induction of xenobiotic metabolizing enzymes: a feedforward control mechanism for potential hormetic responses.

    PubMed

    Zhang, Qiang; Pi, Jingbo; Woods, Courtney G; Andersen, Melvin E

    2009-06-15

    Hormetic responses to xenobiotic exposure likely occur as a result of overcompensation by the homeostatic control systems operating in biological organisms. However, the mechanisms underlying overcompensation that leads to hormesis are still unclear. A well-known homeostatic circuit in the cell is the gene induction network comprising phase I, II and III metabolizing enzymes, which are responsible for xenobiotic detoxification, and in many cases, bioactivation. By formulating a differential equation-based computational model, we investigated in this study whether hormesis can arise from the operation of this gene/enzyme network. The model consists of two feedback and one feedforward controls. With the phase I negative feedback control, xenobiotic X activates nuclear receptors to induce cytochrome P450 enzyme, which bioactivates X into a reactive metabolite X'. With the phase II negative feedback control, X' activates transcription factor Nrf2 to induce phase II enzymes such as glutathione S-transferase and glutamate cysteine ligase, etc., which participate in a set of reactions that lead to the metabolism of X' into a less toxic conjugate X''. The feedforward control involves phase I to II cross-induction, in which the parent chemical X can also induce phase II enzymes directly through the nuclear receptor and indirectly through transcriptionally upregulating Nrf2. As a result of the active feedforward control, a steady-state hormetic relationship readily arises between the concentrations of the reactive metabolite X' and the extracellular parent chemical X to which the cell is exposed. The shape of dose-response evolves over time from initially monotonically increasing to J-shaped at the final steady state-a temporal sequence consistent with adaptation-mediated hormesis. The magnitude of the hormetic response is enhanced by increases in the feedforward gain, but attenuated by increases in the bioactivation or phase II feedback loop gains. Our study suggests a

  20. Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

    PubMed

    Bell, Stephen A; Niehaus, Thomas D; Nybo, S Eric; Chappell, Joseph

    2014-12-09

    Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

  1. Invertase SUC2 Is the key hydrolase for inulin degradation in Saccharomyces cerevisiae.

    PubMed

    Wang, Shi-An; Li, Fu-Li

    2013-01-01

    Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.

  2. Key Response Planning Factors for the Aftermath of Nuclear Terrorism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Buddemeier, B R; Dillon, M B

    Despite hundreds of above-ground nuclear tests and data gathered from Hiroshima and Nagasaki, the effects of a ground-level, low-yield nuclear detonation in a modern urban environment are still the subject of considerable scientific debate. Extensive review of nuclear weapon effects studies and discussions with nuclear weapon effects experts from various federal agencies, national laboratories, and technical organizations have identified key issues and bounded some of the unknowns required to support response planning for a low-yield, ground-level nuclear detonation in a modern U.S. city. This study, which is focused primarily upon the hazards posed by radioactive fallout, used detailed fallout predictionsmore » from the advanced suite of three-dimensional (3-D) meteorology and plume/fallout models developed at Lawrence Livermore National Laboratory (LLNL), including extensive global Key Response Planning Factors for the Aftermath of Nuclear Terrorism geographical and real-time meteorological databases to support model calculations. This 3-D modeling system provides detailed simulations that account for complex meteorology and terrain effects. The results of initial modeling and analysis were presented to federal, state, and local working groups to obtain critical, broad-based review and feedback on strategy and messaging. This effort involved a diverse set of communities, including New York City, National Capitol Regions, Charlotte, Houston, Portland, and Los Angeles. The largest potential for reducing casualties during the post-detonation response phase comes from reducing exposure to fallout radiation. This can be accomplished through early, adequate sheltering followed by informed, delayed evacuation.B The response challenges to a nuclear detonation must be solved through multiple approaches of public education, planning, and rapid response actions. Because the successful response will require extensive coordination of a large number of organizations

  3. Immunogold Localization of Key Metabolic Enzymes in the Anammoxosome and on the Tubule-Like Structures of Kuenenia stuttgartiensis.

    PubMed

    de Almeida, Naomi M; Neumann, Sarah; Mesman, Rob J; Ferousi, Christina; Keltjens, Jan T; Jetten, Mike S M; Kartal, Boran; van Niftrik, Laura

    2015-07-01

    Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite as the terminal electron acceptor to form dinitrogen gas in the absence of oxygen. Anammox bacteria have a compartmentalized cell plan with a central membrane-bound "prokaryotic organelle" called the anammoxosome. The anammoxosome occupies most of the cell volume, has a curved membrane, and contains conspicuous tubule-like structures of unknown identity and function. It was suggested previously that the catalytic reactions of the anammox pathway occur in the anammoxosome, and that proton motive force was established across its membrane. Here, we used antibodies raised against five key enzymes of the anammox catabolism to determine their cellular location. The antibodies were raised against purified native hydroxylamine oxidoreductase-like protein kustc0458 with its redox partner kustc0457, hydrazine dehydrogenase (HDH; kustc0694), hydroxylamine oxidase (HOX; kustc1061), nitrite oxidoreductase (NXR; kustd1700/03/04), and hydrazine synthase (HZS; kuste2859-61) of the anammox bacterium Kuenenia stuttgartiensis. We determined that all five protein complexes were exclusively located inside the anammoxosome matrix. Four of the protein complexes did not appear to form higher-order protein organizations. However, the present data indicated for the first time that NXR is part of the tubule-like structures, which may stretch the whole length of the anammoxosome. These findings support the anammoxosome as the locus of catabolic reactions of the anammox pathway. Anammox bacteria are environmentally relevant microorganisms that contribute significantly to the release of fixed nitrogen in nature. Furthermore, the anammox process is applied for nitrogen removal from wastewater as an environment-friendly and cost-effective technology. These microorganisms feature a unique cellular organelle, the anammoxosome, which was proposed to contain the energy metabolism of the cell and tubule-like structures with

  4. Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets.

    PubMed

    Basmacioğlu Malayoğlu, H; Baysal, S; Misirlioğlu, Z; Polat, M; Yilmaz, H; Turan, N

    2010-02-01

    1. The study was conducted to determine the effects of dietary supplementation of enzyme and oregano essential oil at two levels, alone or together, on performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal based diets. 2. The following dietary treatments were used from d 0 to 21. Diet 1 (control, CONT): a commercial diet containing no enzyme or oregano essential oil, diet 2 (ENZY): supplemented with enzyme, diet 3 (EO250): supplemented with essential oil at 250 mg/kg feed, diet 4 (EO500): supplemented with essential oil at 500 mg/kg feed, diet 5 (ENZY + EO250): supplemented with enzyme and essential oil at 250 mg/kg, and diet 6 (ENZY + EO500): supplemented with enzyme and essential oil at 500 mg/kg. 3. Birds fed on diets containing ENZY, EO250 and ENZY + EO250 had significantly higher weight gain than those given CONT diet from d 0 to 7. No significant effects on feed intake, feed conversion ratio, mortality, organ weights except for jejunum weight and intestinal lengths was found with either enzyme or essential oil, alone or in combination, over the 21-d growth period. The supplementation of essential oil together with enzyme decreased jejunum weight compared with essential oil alone. 4. Supplementation with enzyme significantly decreased viscosity and increased dry matter of digesta, but did not alter pH of digesta. There was no effect of essential oil alone at either concentration on viscosity, dry matter or pH of digesta. A significant decrease in viscosity of digesta appeared when essential oil was used with together enzyme. 5. The supplementation of essential oil at both levels with or without enzyme significantly increased chymotrypsin activity in the digestive system, and improved crude protein digestibility. 6. The higher concentration of essential oil with and without enzyme significantly increased serum total cholesterol concentrations. No significant effect on immune response

  5. In vitro and in silico Studies of Mangiferin from Aphloia theiformis on Key Enzymes Linked to Diabetes Type 2 and Associated Complications.

    PubMed

    Picot, Marie C N; Zengin, Gokhan; Mollica, Adriano; Stefanucci, Azzurra; Carradori, Simone; Mahomoodally, Mohamad F

    2017-01-01

    Mangiferin, was identified in the crude methanol extract, ethyl acetate, and n-butanol fractions of Aphloia theiformis (Vahl.) Benn. This study aimed to analyze the plausible binding modes of mangiferin to key enzymes linked to diabetes type 2 (DT2), obesity, hypertension, Alzheimer's disease, and urolithiasis using molecular docking. Crystallographic structures of α-amylase, α-glucosidase, glycogen phosphorylase (GP), pancreatic lipase, cholesterol esterase (CEase), angiotensin-I-converting enzyme (ACE), acetyl cholinesterase (AChE), and urease available on the Protein Databank database were docked to mangiferin using Gold 6.0 software. We showed that mangiferin bound to all enzymes by π-π and hydrogen bonds mostly. Mangiferin was docked to both allosteric and orthosteric sites of α-glucosidase by π-π interactions. However, several hydrogen bonds were observed at the orthosteric position, suggesting a preference for this site. The docking of mangiferin on AChE with the catalytic pocket occupied by paraoxon could be attributed to π-π stacking involving amino acid residues, Trp341 and Trp124. This study provided an insight of the molecular interaction of mangiferin with the studied enzymes and can be considered as a valuable tool for designing new drugs for better management of these diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Indicators: Sediment Enzymes

    EPA Pesticide Factsheets

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

  7. Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

    PubMed

    Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

    2016-08-15

    The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the

  8. Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

    PubMed

    Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A

    2015-01-01

    Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

  9. Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

    PubMed

    Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

    2004-12-01

    Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

  10. The Structure of the Flavoprotein Tryptophan-2-Monooxygenase, a Key Enzyme in the Formation of Galls in Plants†

    PubMed Central

    Gaweska, Helena M.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

    2013-01-01

    The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic-acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from Pseudomonas savastanoi has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as the monoamine oxidase family of flavoproteins, with the greatest similarities to the L-amino acid oxidases. The location of bound indole-3-acetamide in the active site enables identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the kcat and kcat/KTrp values by 60,000 and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon-hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the kcat value less than 2-fold and the kcat/KTrp value only 5-fold, while the W466M mutation results in enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin binding site in the more conserved FAD domain. PMID:23521653

  11. Enzyme-Linked Immunosorbent Assay and Serologic Responses to Pneumocystis jiroveci

    PubMed Central

    Koch, Judy; Levin, Linda; Walzer, Peter D.

    2004-01-01

    Seroepidemiologic studies of Pneumocystis pneumonia (PCP) in humans have been limited by inadequate reagents. We have developed an enzyme-linked immunosorbent assay (ELISA) using three overlapping recombinant fragments of the human Pneumocystis major surface glycoprotein (MsgA, MsgB, and MsgC) for analysis of antibody responses in HIV-positive patients and healthy blood donors. HIV-positive patients had significantly higher antibody levels to all Msg fragments. Furthermore, HIV-positive patients who experienced a previous episode of PCP (PCP-positive) had higher level of antibodies to MsgC than patients who never had PCP. A significant association was found between ELISA antibody level and reactivity by Western blot in HIV-positive patients, especially those who were PCP-positive. Thus, this ELISA will be useful in studying serum antibody responses to Pneumocystis in different human populations. PMID:15200818

  12. Effects of dietary inclusion level of a mixture of probiotic cultures and enzymes on broiler chickens immunity response.

    PubMed

    Seidavi, Alireza; Dadashbeiki, Mohammad; Alimohammadi-Saraei, Mohammad-Hossein; van den Hoven, René; Payan-Carreira, Rita; Laudadio, Vito; Tufarelli, Vincenzo

    2017-02-01

    The current study was conducted to analyse the effect of a mixture of probiotic cultures and enzymes (Probio Enzyme, XVET GmbH, Germany) on the immune response and weight of central lymphoid organs and liver in broilers. A total of 270 male chickens were randomly divided into nine groups, with three replicates of 10 birds each. Treatment groups were fed for either 22 or 42 days with different levels of Probio Enzyme 250, 500, 750 and 1000 g/ton, whereas the control group fed a basal diet without Probio Enzyme. To analyse the effects of dietary supplementation on broilers humoral immune response, the antibodies titres for avian influenza (AI) and Newcastle disease virus (NDV) and to the sheep red blood cells (SRBC) challenge were assayed in birds from each experimental unit, along with the assessment of the weight of the main lymphoid organs and liver. The addition of the Probio Enzyme mixture did not significantly affect the titres of the antibodies against AI and NDV at day 42, despite the wide individual variation observed specially on the antibody titres at day 33. Treatments affected the production of IgG after the second challenge with SRBC (P = 0.003), which was transposed to the correspondent total Ig titres (P = 0.044). Conversely, a lower birds' body weight (BW) was found in the majority of treated groups compared to control (P = 0.031). The spleen was the only lymphoid organ showing differences in the absolute and relative weight (P = 0.003 and P = 0.001, respectively). No differences were found in thymus and Bursa of Fabricius weights. In conclusion, broilers treated with Probio Enzyme showed a satisfactory immune response compared with control, despite the wide variation found after the first vaccine challenge against AI. Moreover, the probiotic mixture dose and duration modulated differently the immune response and the spleen weight, unaffecting the central lymphoid organs weight.

  13. New Stable Isotope Method to Measure Protein Digestibility and Response to Pancreatic Enzyme Intake in Cystic Fibrosis

    PubMed Central

    Engelen, M.P.K.J.; Com, G.; Anderson, P.J.; Deutz, N.E.P.

    2015-01-01

    Background & Aims Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. Objective To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. Design In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 hours by adding 15N-labeled spirulina protein and L-[ring-2H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [15N]PHE to [2H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-13C-5,5-2H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Results Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P<0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n=10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Conclusion Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. PMID:24268783

  14. Virulence-Associated Enzymes of Cryptococcus neoformans

    PubMed Central

    Almeida, Fausto; Wolf, Julie M.

    2015-01-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

  15. Droplet-based microfluidics for dose-response assay of enzyme inhibitors by electrochemical method.

    PubMed

    Gu, Shuqing; Lu, Youlan; Ding, Yaping; Li, Li; Zhang, Fenfen; Wu, Qingsheng

    2013-09-24

    A simple but robust droplet-based microfluidic system was developed for dose-response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose-response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC50 (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. New stable isotope method to measure protein digestibility and response to pancreatic enzyme intake in cystic fibrosis.

    PubMed

    Engelen, M P K J; Com, G; Anderson, P J; Deutz, N E P

    2014-12-01

    Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 h by adding (15)N-labeled spirulina protein and L-[ring-(2)H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [(15)N]PHE to [(2)H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-(13)C-5,5-(2)H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P < 0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n = 10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. Registration ClinicalTrials.gov = NCT01494909. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  17. Nicotine affects rat Leydig cell function in vivo and vitro via down-regulating some key steroidogenic enzyme expressions.

    PubMed

    Guo, Xiaoling; Wang, Huang; Wu, Xiaolong; Chen, Xianwu; Chen, Yong; Guo, Jingjing; Li, Xiaoheng; Lian, Qingquan; Ge, Ren-Shan

    2017-12-01

    Nicotine is consumed largely as a component of cigarettes and has a potential effect on pubertal development of Leydig cells in males. To investigate its effects, 49-day-old male Sprague Dawley rats received intraperitoneal injections of nicotine (0.5 or 1 mg/kg/day) for 2 weeks and immature Leydig cells were isolated from the testes of 35-day-old rats and treated with nicotine (0.05-50 μM). Serum hormones, Leydig cell number and related gene expression levels after in vivo treatment were determined and medium androgen levels were measured and cell cycle, apoptosis, mitochondrial membrane potential (△Ψm), and reactive oxygen species (ROS) of Leydig cells after in vitro treatment were measured. In vivo exposure to nicotine lowered serum luteinizing hormone, follicle stimulating hormone, and testosterone levels and reduced Leydig cell number and gene expression levels. Nicotine in vitro inhibited androgen production in Leydig cells by downregulating the expression levels of P450 cholesterol side cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, and steroidogenic factor 1 at different concentration ranges. In conclusion, nicotine disrupts Leydig cell steroidogenesis during puberty possibly via down-regulating some key steroidogenic enzyme expressions. Copyright © 2017. Published by Elsevier Ltd.

  18. Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences.

    PubMed

    Schwarzenberger, Anke; Fink, Patrick

    2018-04-01

    Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and β-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Response of oxidative enzyme activities to nitrogen deposition affects soil concentrations of dissolved organic carbon

    USGS Publications Warehouse

    Waldrop, M.P.; Zak, D.R.

    2006-01-01

    Recent evidence suggests that atmospheric nitrate (NO3- ) deposition can alter soil carbon (C) storage by directly affecting the activity of lignin-degrading soil fungi. In a laboratory experiment, we studied the direct influence of increasing soil NO 3- concentration on microbial C cycling in three different ecosystems: black oak-white oak (BOWO), sugar maple-red oak (SMRO), and sugar maple-basswood (SMBW). These ecosystems span a broad range of litter biochemistry and recalcitrance; the BOWO ecosystem contains the highest litter lignin content, SMRO had intermediate lignin content, and SMBW leaf litter has the lowest lignin content. We hypothesized that increasing soil solution NO 3- would reduce lignolytic activity in the BOWO ecosystem, due to a high abundance of white-rot fungi and lignin-rich leaf litter. Due to the low lignin content of litter in the SMBW, we further reasoned that the NO3- repression of lignolytic activity would be less dramatic due to a lower relative abundance of white-rot basidiomycetes; the response in the SMRO ecosystem should be intermediate. We increased soil solution NO3- concentrations in a 73-day laboratory incubation and measured microbial respiration and soil solution dissolved organic carbon (DOC) and phenolics concentrations. At the end of the incubation, we measured the activity of ??-glucosidase, N-acetyl-glucosaminidase, phenol oxidase, and peroxidase, which are extracellular enzymes involved with cellulose and lignin degradation. We quantified the fungal biomass, and we also used fungal ribosomal intergenic spacer analysis (RISA) to gain insight into fungal community composition. In the BOWO ecosystem, increasing NO 3- significantly decreased oxidative enzyme activities (-30% to -54%) and increased DOC (+32% upper limit) and phenolic (+77% upper limit) concentrations. In the SMRO ecosystem, we observed a significant decrease in phenol oxidase activity (-73% lower limit) and an increase in soluble phenolic concentrations

  20. Response surface optimization of the substance colour indigo production by amylase enzyme

    NASA Astrophysics Data System (ADS)

    Handayani, Prima Astuti; Megawati, Kusdianto, Nugraha, Deny Aditia; Novitasari, Lilis

    2017-03-01

    Indigofera leaf production in Indonesia reaches 30 tons of dry matter per hectare per year. Indigo which produce exclusive blue colour already used to dyeing textile, specially "Batik". Batik cloth using natural dyes has artistic value and distinctive colours, as well as ethnic and exclusive impression that have a high value. Indigofera leaves containing blue dye that can be obtained through hydrolysis and oxidation. The hydrolysis reaction using enzyme catalyst. The research objective is to obtain optimum operating conditions of the hydrolysis reaction in the extraction of blue dye with a cellulase enzyme catalyst. Indigofera used leaves 5 month old and tools used include reactors, stirrer, aerator, autoclaves, incubators and ovens. Optimization parameters are studied an α-amylase enzyme concentration of 2.5-10 wt%, pH 5-9 and a reaction time of 4-10 days. The concentration of blue dye was analyzed by gravimetric method. Experimental data were analyzed by the method of Response Surface Methodology and central composite design, the model corresponding linear model with a mathematical equation Y = 6.22763 - 0.02584X1 - 1.25889X2 - 0.42239X3+0.00694X12+ 0.08872X22+ 0.03747X32+ 0.01372X1X2 -0.00582X1X3 - 0.00208X2X3 The optimum operating conditions in the range of studied enzym concentration of 3.1 wt%, pH 7.4 and the hydrolysis reaction time of 5.6 days with a yield dye of 1,42 %.

  1. Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD)

    PubMed Central

    Piontkivska, Helen; Chung, J. Sook; Ivanina, Anna V.; Sokolov, Eugene P.; Techa, Sirinart; Sokolova, Inna M.

    2010-01-01

    Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O2- and Fe2+-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional

  2. Etiological classification of depression based on the enzymes of tryptophan metabolism.

    PubMed

    Fukuda, Katsuhiko

    2014-12-24

    Viewed in terms of input and output, the mechanisms of depression are still akin to a black box. However, there must be main pivots for diverse types of depression. From recent therapeutic observations, both the serotonin (5-HT) and kynurenine pathways of tryptophan metabolism may be of particular importance to improved understanding of depression. Here, I propose an etiological classification of depression, based on key peripheral and central enzymes of tryptophan metabolism. Endogenous depression is caused by a larger genetic component than reactive depression. Besides enterochromaffin and mast cells, tryptophan hydroxylase 1 (TPH1), primarily expressed in the gastrointestinal tract, is also found in 5-hydroxytryptophan-producing cells (5-HTP cells) in normal intestinal enterocytes, which are thought to essentially shunt 5-HT production in 5-HT-producing cells. Genetic studies have reported an association between TPH1 and depression, or the responsiveness of depression to antidepressive medication. Therefore, it is possible that hypofunctional 5-HTP cells (reflecting TPH1 dysfunction) in the periphery lead to deficient brain 5-HT levels. Additionally,it has been reported that higher TPH2 expression in depressed suicides may reflect a homeostatic response to deficient 5-HT levels. Subsequently, endogenous depression may be caused by TPH1 dysfunction combined with compensatory TPH2 activation. Reactive depression results from life stresses and involves the hypothalamic-pituitary-adrenal axis, with resulting cortisol production inducing tryptophan 2,3-dioxygenase (TDO) activation. In secondary depression, caused by inflammation, infection, or oxidative stress, indoleamine 2,3-dioxygenase (IDO) is activated. In both reactive and secondary depression, the balance between 3-hydroxykynurenine (3-HK) and kynurenic acid may shift towards 3-HK production via kynurenine-3-monooxygenase (KMO) activation. By shifting the equilibrium position of key enzymes of tryptophan

  3. Enzyme and Thermal Dual Responsive Amphiphilic Polymer Core-Shell Nanoparticle for Doxorubicin Delivery to Cancer Cells.

    PubMed

    Kashyap, Smita; Singh, Nitesh; Surnar, Bapurao; Jayakannan, Manickam

    2016-01-11

    Dual responsive polymer nanoscaffolds for administering anticancer drugs both at the tumor site and intracellular compartments are made for improving treatment in cancers. The present work reports the design and development of new thermo- and enzyme-responsive amphiphilic copolymer core-shell nanoparticles for doxorubicin delivery at extracellular and intracellular compartments, respectively. A hydrophobic acrylate monomer was tailor-made from 3-pentadecylphenol (PDP, a natural resource) and copolymerized with oligoethylene glycol acrylate (as a hydrophilic monomer) to make new classes of thermo and enzyme dual responsive polymeric amphiphiles. Both radical and reversible addition-fragmentation chain transfer (RAFT) methodologies were adapted for making the amphiphilic copolymers. These amphiphilic copolymers were self-assembled to produce spherical core-shell nanoparticles in water. Upon heating, the core-shell nanoparticles underwent segregation to produce larger sized aggregates above the lower critical solution temperature (LCST). The dual responsive polymer scaffold was found to be capable of loading water insoluble drug, such as doxorubicin (DOX), and fluorescent probe-like Nile Red. The drug release kinetics revealed that DOX was preserved in the core-shell assemblies at normal body temperature (below LCST, ≤ 37 °C). At closer to cancer tissue temperature (above LCST, ∼43 °C), the polymeric scaffold underwent burst release to deliver 90% of loaded drugs within 2 h. At the intracellular environment (pH 7.4, 37 °C) in the presence of esterase enzyme, the amphiphilic copolymer ruptured in a slow and controlled manner to release >95% of the drugs in 12 h. Thus, both burst release of cargo at the tumor microenvironment and control delivery at intracellular compartments were accomplished in a single polymer scaffold. Cytotoxicity assays of the nascent and DOX-loaded polymer were carried out in breast cancer (MCF-7) and cervical cancer (HeLa) cells. Among

  4. Role of conformational dynamics in the evolution of novel enzyme function.

    PubMed

    Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

    2018-05-21

    The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

  5. Virus scaffolds as enzyme nano-carriers.

    PubMed

    Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

    2012-07-01

    The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

    PubMed

    Shlaifer, Irina; Turnbull, Joanne L

    2016-07-01

    Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.

  7. Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal.

    PubMed

    Kohl, Kevin D; Dearing, M Denise

    2011-12-15

    Many plants produce plant secondary compounds (PSCs) that bind and inhibit the digestive enzymes of herbivores, thus limiting digestibility for the herbivore. Herbivorous insects employ several physiological responses to overcome the anti-nutritive effects of PSCs. However, studies in vertebrates have not shown such responses, perhaps stemming from the fact that previously studied vertebrates were not herbivorous. The responses of the digestive system to dietary PSCs in populations of Bryant's woodrat (Neotoma bryanti) that vary in their ecological and evolutionary experience with the PSCs in creosote bush (Larrea tridentata) were compared. Individuals from naïve and experienced populations were fed diets with and without added creosote resin. Animals fed diets with creosote resin had higher activities of pancreatic amylase, as well as luminal amylase and chymotrypsin, regardless of prior experience with creosote. The experienced population showed constitutively higher activities of intestinal maltase and sucrase. Additionally, the naïve population produced an aminopeptidase-N enzyme that was less inhibited by creosote resin when feeding on the creosote resin diet, whereas the experienced population constitutively expressed this form of aminopeptidase-N. Thus, the digestive system of an herbivorous vertebrate responds significantly to dietary PSCs, which may be important for allowing herbivorous vertebrates to feed on PSC-rich diets.

  8. Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes

    PubMed Central

    Chervin, Stephanie M.; Kittendorf, Jeffrey D.; Garcia, George A.

    2009-01-01

    Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT). PMID:17673081

  9. Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

    NASA Astrophysics Data System (ADS)

    Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

    2015-12-01

    Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly

  10. A Deubiquitinating Enzyme Ubp14 Is Required for Development, Stress Response, Nutrient Utilization, and Pathogenesis of Magnaporthe oryzae.

    PubMed

    Wang, Zhao; Zhang, Hong; Liu, Caiyun; Xing, Junjie; Chen, Xiao-Lin

    2018-01-01

    Ubiquitination is an essential protein modification in eukaryotic cells, which is reversible. Deubiquitinating enzymes (DUBs) catalyze deubiquitination process to reverse ubiquitination, maintain ubiquitin homeostasis or promote protein degradation by recycling ubiquitins. In order to investigate effects of deubiquitination process in plant pathogenic fungus Magnaporthe oryzae , we generated deletion mutants of MoUBP14 . Ortholog of MoUbp14 was reported to play general roles in ubiquitin-mediated protein degradation in Saccharomyces cerevisiae . The Δ Moubp14 mutant lost its pathogenicity and was severely reduced in mycelial growth, sporulation, carbon source utilization, and increased in sensitivity to distinct stresses. The mutant was blocked in penetration, which could due to defect in turgor generation. It is also blocked in invasive growth, which could due to reduction in stress tolerance and nutrient utilization. Deletion of UBP14 also led to accumulation of free polyubiquitin chains. Pulldown assay identified some proteins related to carbohydrate metabolism and stress response may putatively interact with MoUbp14, including two key rate-limiting enzymes of gluconeogenesis, MoFbp1 and MoPck1. These two proteins were degraded when the glucose was supplied to M. oryzae grown in low glucose media for a short period of time (∼12 h), and this process required MoUbp14. In summary, pleiotropic phenotypes of the deletion mutants indicated that MoUbp14 is required for different developments and pathogenicity of M. oryzae .

  11. In vivo phosphoproteome characterization reveals key starch granule-binding phosphoproteins involved in wheat water-deficit response.

    PubMed

    Chen, Guan-Xing; Zhen, Shou-Min; Liu, Yan-Lin; Yan, Xing; Zhang, Ming; Yan, Yue-Ming

    2017-10-23

    Drought stress during grain development causes significant yield loss in cereal production. The phosphorylated modification of starch granule-binding proteins (SGBPs) is an important mechanism regulating wheat starch biosynthesis. In this study, we performed the first proteomics and phosphoproteomics analyses of SGBPs in elite Chinese bread wheat (Triticum aestivum L.) cultivar Jingdong 17 under well-watered and water-stress conditions. Water stress treatment caused significant reductions in spike grain numbers and weight, total starch and amylopectin content, and grain yield. Two-dimensional gel electrophoresis revealed that the quantity of SGBPs was reduced significantly by water-deficit treatment. Phosphoproteome characterization of SGBPs under water-deficit treatment demonstrated a reduced level of phosphorylation of main starch synthesis enzymes, particularly for granule-bound starch synthase (GBSS I), starch synthase II-a (SS II-a), and starch synthase III (SS III). Specifically, the Ser34 site of the GBSSI protein, the Tyr358 site of SS II-a, and the Ser837 site of SS III-a exhibited significant less phosphorylation under water-deficit treatment than well-watered treatment. Furthermore, the expression levels of several key genes related with starch biosynthesis detected by qRT-PCR were decreased significantly at 15 days post-anthesis under water-deficit treatment. Immunolocalization showed a clear movement of GBSS I from the periphery to the interior of starch granules during grain development, under both water-deficit and well-watered conditions. Our results demonstrated that the reduction in gene expression or transcription level, protein expression and phosphorylation levels of starch biosynthesis related enzymes under water-deficit conditions is responsible for the significant decrease in total starch content and grain yield.

  12. Glutathione-related enzymes and the eye.

    PubMed

    Ganea, Elena; Harding, John J

    2006-01-01

    Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and

  13. Post Outbreak Review: Dengue Preparedness and Response in Key West, Florida

    PubMed Central

    Hayden, Mary H.; Cavanaugh, Jamie L.; Tittel, Christopher; Butterworth, Melinda; Haenchen, Steven; Dickinson, Katherine; Monaghan, Andrew J.; Ernst, Kacey C.

    2015-01-01

    Dengue is the most prevalent mosquito-borne viral infection. Recent outbreaks in the southern United States illustrate the risk of reemergence. The first autochthonous cases since 1934 in Key West, FL, occurred in 2009–2010. We conducted a survey in 2012 with decision makers instrumental to the control of the outbreak to 1) determine their awareness of the multiple strategies used to control the outbreak and 2) assess their perceptions of the relative effectiveness of these strategies. An online survey was delivered to a predefined list of decision makers from multiple sectors to better understand dengue preparedness and response. Thirty-six out of 45 surveys were returned for an 80% response rate. Results indicate the need to focus prevention strategies on educational campaigns designed to increase population awareness of transmission risk. Respondents remain concerned about future dengue transmission risk in Key West and lack of resources to respond. PMID:26078319

  14. An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

    PubMed

    Eslami, Habib; Mohtashami, Seyed Kaveh; Basmanj, Maryam Taghavi; Rahati, Maryam; Rahimi, Hamzeh

    2017-07-01

    The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg 2+ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg 2+ . Such detailed understanding will probably help to design an improved enzyme.

  15. 2008 GRC Iron Sulfur Enzymes-Conference to be held June 8-13, 2008

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cramer, Stephen; Gray, Nancy Ryan

    2009-01-01

    Iron-sulfur proteins are among the most common and ancient enzymes and electron-transfer agents in nature. They play key roles in photosynthesis, respiration, and the metabolism of small molecules such as H2, CO, and N2. The Iron Sulfur Enzyme Gordon Research Conference evolved from an earlier GRC on Nitrogen Fixation that began in 1994. The scope of the current meeting has broadened to include all enzymes or metalloproteins in which Fe-S bonds play a key role. This year's meeting will focus on the biosynthesis of Fe-S clusters, as well as the structure and mechanism of key Fe-S enzymes such as hydrogenase,more » nitrogenase and its homologues, radical SAM enzymes, and aconitase-related enzymes. Recent progress on the role of Fe-S enzymes in health, disease, DNA/RNA-processing, and alternative bio-energy systems will also be highlighted. This conference will assemble a broad, diverse, and international group of biologists and chemists who are investigating fundamental issues related to Fe-S enzymes, on atomic, molecular, organism, and environmental scales. The topics to be addressed will include: Biosynthesis & Genomics of Fe-S Enzymes; Fundamental Fe-S Chemistry; Hydrogen and Fe-S Enzymes; Nitrogenase & Homologous Fe-S Enzymes; Fe-S Enzymes in Health & Disease; Radical SAM and Aconitase-Related Fe-S Enzymes; Fe-S Enzymes and Synthetic Analogues in BioEnergy; and Fe-S Enzymes in Geochemistry and the Origin of Life.« less

  16. Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.

    PubMed

    Doi, Hidetaka; Tokura, Yuriko; Mori, Yukiko; Mori, Kenichi; Asakura, Yoko; Usuda, Yoshihiro; Fukuda, Hiroo; Chinen, Akito

    2017-02-01

    Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2 T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

  17. Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

    PubMed Central

    Zharikova, Olga L.; Fokina, Valentina M.; Nanovskaya, Tatiana N.; Hill, Ronald A.; Mattison, Donald R.; Hankins, Gary D.V.; Ahmed, Mahmoud S.

    2014-01-01

    One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans- (M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a( 4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo. PMID:19679108

  18. Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide.

    PubMed

    Zharikova, Olga L; Fokina, Valentina M; Nanovskaya, Tatiana N; Hill, Ronald A; Mattison, Donald R; Hankins, Gary D V; Ahmed, Mahmoud S

    2009-12-15

    One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

  19. Suppression of 9-cis-epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato.

    PubMed

    Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

    2012-01-01

    Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).

  20. Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes.

    PubMed

    Merz, Michael; Eisele, Thomas; Berends, Pieter; Appel, Daniel; Rabe, Swen; Blank, Imre; Stressler, Timo; Fischer, Lutz

    2015-06-17

    Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

  1. Post Outbreak Review: Dengue Preparedness and Response in Key West, Florida.

    PubMed

    Hayden, Mary H; Cavanaugh, Jamie L; Tittel, Christopher; Butterworth, Melinda; Haenchen, Steven; Dickinson, Katherine; Monaghan, Andrew J; Ernst, Kacey C

    2015-08-01

    Dengue is the most prevalent mosquito-borne viral infection. Recent outbreaks in the southern United States illustrate the risk of reemergence. The first autochthonous cases since 1934 in Key West, FL, occurred in 2009-2010. We conducted a survey in 2012 with decision makers instrumental to the control of the outbreak to 1) determine their awareness of the multiple strategies used to control the outbreak and 2) assess their perceptions of the relative effectiveness of these strategies. An online survey was delivered to a predefined list of decision makers from multiple sectors to better understand dengue preparedness and response. Thirty-six out of 45 surveys were returned for an 80% response rate. Results indicate the need to focus prevention strategies on educational campaigns designed to increase population awareness of transmission risk. Respondents remain concerned about future dengue transmission risk in Key West and lack of resources to respond. © The American Society of Tropical Medicine and Hygiene.

  2. In vitro inhibitory potential of Cynara scolymus, Silybum marianum, Taraxacum officinale, and Peumus boldus on key enzymes relevant to metabolic syndrome.

    PubMed

    Villiger, Angela; Sala, Filippo; Suter, Andy; Butterweck, Veronika

    2015-01-15

    Boldocynara®, a proprietary dietary supplement product consisting of the plants Cynara scolymus, Silybum marianum, Taraxacum officinale, and Peumus boldus, used to promote functions of the liver and the gallbladder. It was the aim of the present study to look from a different perspective at the product by investigating the in vitro potential of Boldocynara® as a combination product and its individual extracts on key enzymes relevant to metabolic syndrome. Peumus boldus extract exhibited pronounced inhibitory activities on α-glucosidase (80% inhibition at 100 µg/ml, IC50: 17.56 µg/ml). Silybum marianum had moderate pancreatic lipase (PL) inhibitory activities (30% at 100 µg/ml) whereas Cynara scolymus showed moderate ACE inhibitory activity (31% at 100 µg/ml). The combination had moderate to weak effects on the tested enzymes. In conclusion, our results indicate some moderate potential of the dietary supplement Boldocynara® and its single ingredients for the prevention of metabolic disorders. Copyright © 2014 Elsevier GmbH. All rights reserved.

  3. Dual enzyme responsive microcapsules simulating an "OR" logic gate for biologically triggered drug delivery applications.

    PubMed

    Radhakrishnan, Krishna; Tripathy, Jasaswini; Raichur, Ashok M

    2013-06-14

    Hollow microcapsules capable of disintegrating in response to dual biological stimuli have been synthesized from two FDA approved drug molecules. The capsules fabricated from protamine and chondroitin sulphate disintegrate in the presence of either trypsin or hyaluronidase enzymes, which are documented to be simultaneously over-expressed under some pathological conditions.

  4. Microbial responses to multi-factor climate change: effects on soil enzymes.

    PubMed

    Steinweg, J Megan; Dukes, Jeffrey S; Paul, Eldor A; Wallenstein, Matthew D

    2013-01-01

    The activities of extracellular enzymes, the proximate agents of decomposition in soils, are known to depend strongly on temperature, but less is known about how they respond to changes in precipitation patterns, and the interaction of these two components of climate change. Both enzyme production and turnover can be affected by changes in temperature and soil moisture, thus it is difficult to predict how enzyme pool size may respond to altered climate. Soils from the Boston-Area Climate Experiment (BACE), which is located in an old field (on abandoned farmland), were used to examine how climate variables affect enzyme activities and microbial biomass carbon (MBC) in different seasons and in soils exposed to a combination of three levels of precipitation treatments (ambient, 150% of ambient during growing season, and 50% of ambient year-round) and four levels of warming treatments (unwarmed to ~4°C above ambient) over the course of a year. Warming, precipitation and season had very little effect on potential enzyme activity. Most models assume that enzyme dynamics follow microbial biomass, because enzyme production should be directly controlled by the size and activity of microbial biomass. We observed differences among seasons and treatments in mass-specific potential enzyme activity, suggesting that this assumption is invalid. In June 2009, mass-specific potential enzyme activity, using chloroform fumigation-extraction MBC, increased with temperature, peaking under medium warming and then declining under the highest warming. This finding suggests that either enzyme production increased with temperature or turnover rates decreased. Increased maintenance costs associated with warming may have resulted in increased mass-specific enzyme activities due to increased nutrient demand. Our research suggests that allocation of resources to enzyme production could be affected by climate-induced changes in microbial efficiency and maintenance costs.

  5. Enzyme activities by indicator of quality in organic soil

    NASA Astrophysics Data System (ADS)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  6. The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

    PubMed Central

    Mannion, Niamh M.; Greenwood, Sam M.; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P.; McLaughlin, Paul J.; Jantsch, Michael F.; Dorin, Julia; Adams, Ian R.; Scadden, A.D.J.; Öhman, Marie; Keegan, Liam P.; O’Connell, Mary A.

    2014-01-01

    Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

  7. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

    PubMed

    Mannion, Niamh M; Greenwood, Sam M; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P; McLaughlin, Paul J; Jantsch, Michael F; Dorin, Julia; Adams, Ian R; Scadden, A D J; Ohman, Marie; Keegan, Liam P; O'Connell, Mary A

    2014-11-20

    The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Self-powered enzyme micropumps

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  9. Modified kinetics of enzymes interacting with nanoparticles

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Breger, Joyce C.; Malanoski, Anthony; Claussen, Jonathan C.; Walper, Scott A.; Ancona, Mario G.; Brown, Carl W.; Stewart, Michael H.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2015-08-01

    Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

  10. Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis).

    PubMed

    Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

    2015-01-01

    Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants' growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., 'Photosynthesis'), GO terms (e.g., 'response to karrikin') and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology.

  11. A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis.

    PubMed

    Jones, Hannah B L; Wells, Stephen A; Prentice, Erica J; Kwok, Anthony; Liang, Liyin L; Arcus, Vickery L; Pudney, Christopher R

    2017-09-01

    Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach. © 2017 Federation of European Biochemical Societies.

  12. Dissipative Dynamics of Enzymes

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

    2014-11-01

    We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C ; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

  13. Dissipative Dynamics of Enzymes

    NASA Astrophysics Data System (ADS)

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni; Zocchi LabMolecular Biophysics Team

    2015-03-01

    We explore enzyme conformational dynamics at sub - Å resolution, specifically temperature effects. The ensemble averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor 2 as the temperature is raised from 10 C to 45 C; the elastic parameter K shows a similar decrease. Thus when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

  14. Dissipative dynamics of enzymes.

    PubMed

    Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

    2014-11-07

    We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

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

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

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

  16. The Key Events Dose-Response Framework: a cross-disciplinary mode-of-action based approach to examining dose-response and thresholds.

    PubMed

    Julien, Elizabeth; Boobis, Alan R; Olin, Stephen S

    2009-09-01

    The ILSI Research Foundation convened a cross-disciplinary working group to examine current approaches for assessing dose-response and identifying safe levels of intake or exposure for four categories of bioactive agents-food allergens, nutrients, pathogenic microorganisms, and environmental chemicals. This effort generated a common analytical framework-the Key Events Dose-Response Framework (KEDRF)-for systematically examining key events that occur between the initial dose of a bioactive agent and the effect of concern. Individual key events are considered with regard to factors that influence the dose-response relationship and factors that underlie variability in that relationship. This approach illuminates the connection between the processes occurring at the level of fundamental biology and the outcomes observed at the individual and population levels. Thus, it promotes an evidence-based approach for using mechanistic data to reduce reliance on default assumptions, to quantify variability, and to better characterize biological thresholds. This paper provides an overview of the KEDRF and introduces a series of four companion papers that illustrate initial application of the approach to a range of bioactive agents.

  17. Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside induced lipid peroxidation in rats' pancreas by phenolic extracts of avocado pear leaves and fruit.

    PubMed

    Oboh, Ganiyu; Isaac, Adelusi Temitope; Akinyemi, Ayodele Jacobson; Ajani, Richard Akinlolu

    2014-09-01

    Persea americana fruit and leaves had been known in folk medicine for their anti-diabetic prowess. Therefore, this study sought to investigate the inhibitory effect of phenolic extract from avocado pear (Persea americana) leaves and fruits on some key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase); and sodium nitroprusside (SNP) induced lipid peroxidation in rats' pancreas in vitro. The phenolic extracts of Persea americana fruit and leaves were extracted using methanol and 1M HCl (1:1 v/v). Thereafter, their inhibitory effects on sodium nitroprusside induced lipid peroxidation and key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) were determined in vitro. The result revealed that the leaves had fruit of avocado pear inhibit both α-amylase and α-glucosidase activities in a dose dependent manner. However, the Peel had the highest α-amylase inhibitory activity while the leaf had the highest α-glucosidase inhibitory activity as revealed by their IC50 value. Furthermore, incubation of the rat pancreas in the presence of 5 mM SNP caused an increase in the malondialdehyde (MDA) content in the tissue, however, introduction of the phenolic extracts inhibited MDA produced in a dose dependent manner. The additive and/or synergistic action of major phenolic compounds such as syringic acid, eugenol, vnillic acid, isoeugenol, guaiacol, kaemferol, catechin, ρ-hydroxybenzoic acid, ferulic acid, apigenin, naringenin, epigallocatechin, epicatechin, lupeol and epigallocatechin-3-O-gallate in avocado pear using gas chromatography (GC) could have contributed to the observed medicinal properties of the plant. Therefore, inhibition of some key enzymes linked to type 2 diabetes and prevention of oxidative stress in the pancreas could be some of the possible mechanism by which they exert their anti-diabetic properties.

  18. Inhibition of Key Enzymes Linked to Type 2 Diabetes and Sodium Nitroprusside Induced Lipid Peroxidation in Rats’ Pancreas by Phenolic Extracts of Avocado Pear Leaves and Fruit

    PubMed Central

    Oboh, Ganiyu; Isaac, Adelusi Temitope; Akinyemi, Ayodele Jacobson; Ajani, Richard Akinlolu

    2014-01-01

    Persea americana fruit and leaves had been known in folk medicine for their anti-diabetic prowess. Therefore, this study sought to investigate the inhibitory effect of phenolic extract from avocado pear (Persea americana) leaves and fruits on some key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase); and sodium nitroprusside (SNP) induced lipid peroxidation in rats’ pancreas in vitro. The phenolic extracts of Persea americana fruit and leaves were extracted using methanol and 1M HCl (1:1 v/v). Thereafter, their inhibitory effects on sodium nitroprusside induced lipid peroxidation and key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) were determined in vitro. The result revealed that the leaves had fruit of avocado pear inhibit both α-amylase and α-glucosidase activities in a dose dependent manner. However, the Peel had the highest α-amylase inhibitory activity while the leaf had the highest α-glucosidase inhibitory activity as revealed by their IC50 value. Furthermore, incubation of the rat pancreas in the presence of 5 mM SNP caused an increase in the malondialdehyde (MDA) content in the tissue, however, introduction of the phenolic extracts inhibited MDA produced in a dose dependent manner. The additive and/or synergistic action of major phenolic compounds such as syringic acid, eugenol, vnillic acid, isoeugenol, guaiacol, kaemferol, catechin, ρ-hydroxybenzoic acid, ferulic acid, apigenin, naringenin, epigallocatechin, epicatechin, lupeol and epigallocatechin-3-O-gallate in avocado pear using gas chromatography (GC) could have contributed to the observed medicinal properties of the plant. Therefore, inhibition of some key enzymes linked to type 2 diabetes and prevention of oxidative stress in the pancreas could be some of the possible mechanism by which they exert their anti-diabetic properties PMID:25324703

  19. Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

    PubMed

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-11-01

    A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

  20. NADP-dependent enzymes are involved in response to salt and hypoosmotic stress in cucumber plants.

    PubMed

    Hýsková, Veronika; Plisková, Veronika; Červený, Václav; Ryšlavá, Helena

    2017-07-01

    Salt stress is one of the most damaging plant stressors, whereas hypoosmotic stress is not considered to be a dangerous type of stress in plants and has been less extensively studied. This study was performed to compare the metabolism of cucumber plants grown in soil with plants transferred to distilled water and to a 100 mM NaCl solution. Even though hypoosmotic stress caused by distilled water did not cause such significant changes in the relative water content, Na+/K+ ratio and Rubisco content as those caused by salt stress, it was accompanied by more pronounced changes in the specific activities of NADP-dependent enzymes. After 3 days, the specific activities of NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves were highest under hypoosmotic stress, and lowest in plants grown in soil. In roots, salt stress caused a decrease in the specific activities of major NADP-enzymes. However, at the beginning of salt stress, NADP-galactose-1-dehydrogenase and ribose-1-dehydrogenase were involved in a plant defense response in both roots and leaves. Therefore, the enhanced demands of NADPH in stress can be replenished by a wide range of NADP-dependent enzymes.

  1. Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway

    PubMed Central

    Yang, Shuping; Li, Xin; Liu, Xinfeng; Ding, Xiangbin; Xin, Xiangbo; Jin, Congfei; Zhang, Sheng; Li, Guangpeng; Guo, Hong

    2018-01-01

    MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN−/−. The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3β pathway and highly consistent with the lower glycogen content in gluteus of MSTN−/−. Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth. PMID:29541418

  2. Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease†

    PubMed Central

    Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E.

    2008-01-01

    Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID:16669630

  3. Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.

    PubMed

    Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E

    2006-05-09

    Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.

  4. Directed evolution: an approach to engineer enzymes.

    PubMed

    Kaur, Jasjeet; Sharma, Rohit

    2006-01-01

    Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

  5. Toward "stable-on-the-table" enzymes: improving key properties of catalase by covalent conjugation with poly(acrylic acid).

    PubMed

    Riccardi, Caterina M; Cole, Kyle S; Benson, Kyle R; Ward, Jessamyn R; Bassett, Kayla M; Zhang, Yiren; Zore, Omkar V; Stromer, Bobbi; Kasi, Rajeswari M; Kumar, Challa V

    2014-08-20

    Several key properties of catalase such as thermal stability, resistance to protease degradation, and resistance to ascorbate inhibition were improved, while retaining its structure and activity, by conjugation to poly(acrylic acid) (PAA, Mw 8000) via carbodiimide chemistry where the amine groups on the protein are appended to the carboxyl groups of the polymer. Catalase conjugation was examined at three different pH values (pH 5.0, 6.0, and 7.0) and at three distinct mole ratios (1:100, 1:500, and 1:1000) of catalase to PAA at each reaction pH. The corresponding products are labeled as Cat-PAA(x)-y, where x is the protein to polymer mole ratio and y is the pH used for the synthesis. The coupling reaction consumed about 60-70% of the primary amines on the catalase; all samples were completely water-soluble and formed nanogels, as evidenced by gel electrophoresis and electron microscopy. The UV circular dichroism (CD) spectra indicated substantial retention of protein secondary structure for all samples, which increased to 100% with increasing pH of the synthesis and polymer mole fraction. Soret CD bands of all samples indicated loss of ∼50% of band intensities, independent of the reaction pH. Catalytic activities of the conjugates increased with increasing synthesis pH, where 55-80% and 90-100% activity was retained for all samples synthesized at pH 5.0 and pH 7.0, respectively, and the Km or Vmax values of Cat-PAA(100)-7 did not differ significantly from those of the free enzyme. All conjugates synthesized at pH 7.0 were thermally stable even when heated to ∼85-90 °C, while native catalase denatured between 55 and 65 °C. All conjugates retained 40-90% of their original activities even after storing for 10 weeks at 8 °C, while unmodified catalase lost all of its activity within 2 weeks, under similar storage conditions. Interestingly, PAA surrounding catalase limited access to the enzyme from large molecules like proteases and significantly increased

  6. Guiding Empirical and Theoretical Explorations of Organic Matter Decay By Synthesizing Temperature Responses of Enzyme Kinetics, Microbes, and Isotope Fluxes

    NASA Astrophysics Data System (ADS)

    Billings, S. A.; Ballantyne, F.; Lehmeier, C.; Min, K.

    2014-12-01

    Soil organic matter (SOM) transformation rates generally increase with temperature, but whether this is realized depends on soil-specific features. To develop predictive models applicable to all soils, we must understand two key, ubiquitous features of SOM transformation: the temperature sensitivity of myriad enzyme-substrate combinations and temperature responses of microbial physiology and metabolism, in isolation from soil-specific conditions. Predicting temperature responses of production of CO2 vs. biomass is also difficult due to soil-specific features: we cannot know the identity of active microbes nor the substrates they employ. We highlight how recent empirical advances describing SOM decay can help develop theoretical tools relevant across diverse spatial and temporal scales. At a molecular level, temperature effects on purified enzyme kinetics reveal distinct temperature sensitivities of decay of diverse SOM substrates. Such data help quantify the influence of microbial adaptations and edaphic conditions on decay, have permitted computation of the relative availability of carbon (C) and nitrogen (N) liberated upon decay, and can be used with recent theoretical advances to predict changes in mass specific respiration rates as microbes maintain biomass C:N with changing temperature. Enhancing system complexity, we can subject microbes to temperature changes while controlling growth rate and without altering substrate availability or identity of the active population, permitting calculation of variables typically inferred in soils: microbial C use efficiency (CUE) and isotopic discrimination during C transformations. Quantified declines in CUE with rising temperature are critical for constraining model CUE estimates, and known changes in δ13C of respired CO2 with temperature is useful for interpreting δ13C-CO2 at diverse scales. We suggest empirical studies important for advancing knowledge of how microbes respond to temperature, and ideas for theoretical

  7. Inhibitory potentials of phenolic-rich extracts from Bridelia ferruginea on two key carbohydrate-metabolizing enzymes and Fe2+-induced pancreatic oxidative stress.

    PubMed

    Afolabi, Olakunle Bamikole; Oloyede, Omotade Ibidun; Agunbiade, Shadrack Oludare

    2018-05-01

    The current study was designed to evaluate the various antioxidant potentials and inhibitory effects of phenolic-rich leaf extracts of Bridelia ferruginea (BF) on the in vitro activities of some key enzymes involved in the metabolism of carbohydrates. In this study, BF leaf free and bound phenolic-rich extracts were used. We quantified total phenolic and flavonoid contents, and evaluated several antioxidant activities using assays for ferric reducing antioxidant power, total antioxidant activity (phosphomolybdenum reducing ability), 1,1-diphenyl-2-picrylhydrazyl and thiobarbituric acid reactive species. Also, extracts were tested for their ability to inhibit α-amylase and α-glucosidase activity. The total phenolic and total flavonoid contents in the free phenolic extract of BF were significantly greater than in the bound phenolic extract. Also, all the antioxidant activities considered were significantly greater in the free phenolic extract than in the bound phenolic extract. In the same vein, the free phenolic-rich extract had a significantly higher percentage inhibition against α-glucosidase activity (IC 50  = 28.5 µg/mL) than the bound phenolic extract (IC 50  = 340.0 µg/mL). On the contrary, the free phenolic extract (IC 50  = 210.0 µg/mL) had significantly lower inhibition against α-amylase than the bound phenolic-rich extract (IC 50  = 190.0 µg/mL). The phenolic-rich extracts of BF leaves showed antioxidant potentials and inhibited two key carbohydrate-metabolizing enzymes in vitro. Copyright © 2018 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.

  8. Application of the photoacoustic method to the measurement of acetylene reduction by nitrogenase enzyme

    NASA Astrophysics Data System (ADS)

    Schramm, D. U.; Sthel, M. S.; Carneiro, L. O.; Franco, A. A.; Campos, A. C.; Vargas, H.

    2005-06-01

    Nitrogenase is an enzyme responsible for the reduction of the atmospheric N2 into NH4^+, which represents the key entry point of the molecular nitrogen into the biogeochemical cycle of nitrogen. This enzyme is present in the rhizobial bacteroids, which are symbionts in a Leguminosae plant (Acacia Holosericea), and also reduces acetylene into ethylene at the same rate as the nitrogen reduction. Therefore, a CO2 Laser Photoacoustic system was used for detecting and monitoring the ethylene emission by the nitrogenase activity, in the rhizobial symbionts in Acacia Holosericea, when they are confined in test tubes with acetylene at two different volumes (0.1 and 0.5 ml). Ethylene concentrations are also determined in the ppm range.

  9. Metabolic enzymes in glial cells of the honeybee brain and their associations with aging, starvation and food response.

    PubMed

    Shah, Ashish K; Kreibich, Claus D; Amdam, Gro V; Münch, Daniel

    2018-01-01

    The honey bee has been extensively studied as a model for neuronal circuit and memory function and more recently has emerged as an unconventional model in biogerontology. Yet, the detailed knowledge of neuronal processing in the honey bee brain contrasts with the very sparse information available on glial cells. In other systems glial cells are involved in nutritional homeostasis, detoxification, and aging. These glial functions have been linked to metabolic enzymes, such as glutamine synthetase and glycogen phosphorylase. As a step in identifying functional roles and potential differences among honey bee glial types, we examined the spatial distribution of these enzymes and asked if enzyme abundance is associated with aging and other processes essential for survival. Using immunohistochemistry and confocal laser microscopy we demonstrate that glutamine synthetase and glycogen phosphorylase are abundant in glia but appear to co-localize with different glial sub-types. The overall spatial distribution of both enzymes was not homogenous and differed markedly between different neuropiles and also within each neuropil. Using semi-quantitative Western blotting we found that rapid aging, typically observed in shortest-lived worker bees (foragers), was associated with declining enzyme levels. Further, we found enzyme abundance changes after severe starvation stress, and that glutamine synthetase is associated with food response. Together, our data indicate that aging and nutritional physiology in bees are linked to glial specific metabolic enzymes. Enzyme specific localization patterns suggest a functional differentiation among identified glial types.

  10. Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

    PubMed

    Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia; Tell, Gianluca

    2014-02-01

    An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

  11. OxyR Is a Key Regulator in Response to Oxidative Stress in Streptomyces avermitilis.

    PubMed

    Liu, Xingchao; Sun, Meng; Cheng, Yaqing; Yang, Renjun; Wen, Ying; Chen, Zhi; Li, Jilun

    2016-02-02

    The role of the H2O2-sensing transcriptional regulator OxyR in oxidative stress responses in Streptomyces avermitilis was investigated. An oxyR deletion mutant was more sensitive to H2O2 and tert-butyl hydroperoxide than was the wild-type strain, indicating that OxyR mediates the defensive system against H2O2 and organic peroxide. Evidence presented herein suggests that in cells treated with exogenous H2O2, the oxidized form of OxyR activated expression of ahpCD by binding to a palindromic sequence of the promoter region. Oxidized OxyR also induced expression of other antioxidant enzymes (KatA1, KatA2, KatA3, OhrB1) and oxidative stress regulators (CatR, OhrR, σR). The thiol-oxidative stress regulator gene sigR was regulated at the transcription level by OxyR. We conclude that OxyR is necessary to activate transcription of sigR from the σR-dependent promoter to express an unstable larger isoform of σR during oxidative stress. In response to oxidative stress, OxyR facilitates rapid production of H2O2-scavenging enzymes to repair oxidative damage through direct regulation and cascaded regulation of CatR, OhrR, and σR.

  12. Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

    PubMed

    Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

    2015-03-01

    Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (p<0.05) higher inhibitory effect on α-amylase [IC50 (concentration of sample causing 50% enzyme inhibition)=3.68 μg/mL] and α-glucosidase (IC50=4.98 μg/mL) activities than chlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

  13. The toxicology and immunology of detergent enzymes.

    PubMed

    Basketter, David; Berg, Ninna; Kruszewski, Francis H; Sarlo, Katherine; Concoby, Beth

    2012-01-01

    Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

  14. Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

    PubMed

    Atack, John M; Srikhanta, Yogitha N; Djoko, Karrera Y; Welch, Jessica P; Hasri, Norain H M; Steichen, Christopher T; Vanden Hoven, Rachel N; Grimmond, Sean M; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

    2013-06-01

    NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

  15. Enzyme reactor design under thermal inactivation.

    PubMed

    Illanes, Andrés; Wilson, Lorena

    2003-01-01

    Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

  16. An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge

    PubMed Central

    Onwubiko, J.; Zaru, R.; Rosanoff, S.; Antunes, R.; Bingley, M.; Watkins, X.; O'Donovan, C.; Martin, M. J.

    2017-01-01

    Abstract Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al., (2013), BMC Bioinformatics, 14 (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al., Nucleic Acids Res.2016; 44, D385–D395) Rhea—a database of enzyme-catalysed reactions (Morgat, A., et al., Nucleic Acids Res. 2015; 43, D459-D464), Reactome—a database of biochemical pathways (Fabregat, A., et al., Nucleic Acids Res. 2016; 44, D481–D487), IntEnz—a resource with enzyme nomenclature information (Fleischmann, A., et al., Nucleic Acids Res. 2004 32, D434–D437) and ChEBI (Hastings, J., et al., Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al., Nucleic Acids Res. 201442, 1083–1090)—resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. PMID:28158609

  17. An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge.

    PubMed

    Pundir, S; Onwubiko, J; Zaru, R; Rosanoff, S; Antunes, R; Bingley, M; Watkins, X; O'Donovan, C; Martin, M J

    2017-03-01

    Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al. , (2013), BMC Bioinformatics , (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al ., Nucleic Acids Res. 2016; , D385-D395) Rhea-a database of enzyme-catalysed reactions (Morgat, A., et al .,  Nucleic Acids Res.  2015; , D459-D464), Reactome-a database of biochemical pathways (Fabregat, A., et al ., Nucleic Acids Res. 2016;  , D481-D487), IntEnz-a resource with enzyme nomenclature information (Fleischmann, A., et al ., Nucleic Acids Res.  2004 , D434-D437) and ChEBI (Hastings, J., et al .,  Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al ., Nucleic Acids Res.  2014 , 1083-1090)-resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  18. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    PubMed

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Ameliorating effect of berbamine on hepatic key enzymes of carbohydrate metabolism in high-fat diet and streptozotocin induced type 2 diabetic rats.

    PubMed

    Sankaranarayanan, Chandrasekaran; Nishanthi, Ramajayam; Pugalendi, Pachaiappan

    2018-07-01

    Aberrations in the activities of key enzymes of carbohydrate metabolism is well documented in diabetes mellitus. Previous studies have shown that active ingredients in the extracts of Berberis aristata exhibits diverse pharmacological activities in animal models. The present study was undertaken to investigate whether berbamine (BBM), an alkaloid from the roots of Berberis aristata can ameliorate the altered activities of carbohydrate metabolic enzymes in high fat diet (HFD)/streptozotocin (STZ) induced diabetic rats. Supplementation of HFD for 4 weeks followed by intraperitonial administration of single low dose of STZ (40 mg/kg b.w.) to Sprague Dawley rats resulted in significant hyperglycemia with a decline in plasma insulin levels. The rats also exhibited decreased hemoglobin with an increase in glycated hemoglobin levels. The activities of hexokinase, glucose-6-phosphate dehydrogenase were decreased whereas increases in the activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase were observed in the hepatic tissues of diabetic control rats. Glycogen content in the hepatic and skeletal muscle tissues were found to be decreased in diabetic rats. Oral administration of BBM for 56 days, dose dependently (50, 100, 200 mg/kg b.w.) improved insulin secretion in diabetic treated rats. Immunohistochemical studies on pancreas revealed a strong immunoreactivity to insulin in BBM treated rats. At the effective dose of 100 mg/kg b.w., BBM restored the altered activities of carbohydrate metabolic enzymes and also improved glycogen content in insulin dependent tissues. From the biochemical and histochemical data obtained in this study we conclude that BBM ameliorated the activities of metabolic enzymes and maintained glucose homeostasis in HFD/STZ induced diabetic rats and it can be used as a potential phytomedicine for the management of diabetes mellitus. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  20. Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains.

    PubMed

    Bragin, E Yu; Shtratnikova, V Yu; Dovbnya, D V; Schelkunov, M I; Pekov, Yu A; Malakho, S G; Egorova, O V; Ivashina, T V; Sokolov, S L; Ashapkin, V V; Donova, M V

    2013-11-01

    A comparative genome analysis of Mycobacterium spp. VKM Ac-1815D, 1816D and 1817D strains used for efficient production of key steroid intermediates (androst-4-ene-3,17-dione, AD, androsta-1,4-diene-3,17-dione, ADD, 9α-hydroxy androst-4-ene-3,17-dione, 9-OH-AD) from phytosterol has been carried out by deep sequencing. The assembled contig sequences were analyzed for the presence putative genes of steroid catabolism pathways. Since 3-ketosteroid-9α-hydroxylases (KSH) and 3-ketosteroid-Δ(1)-dehydrogenase (Δ(1) KSTD) play key role in steroid core oxidation, special attention was paid to the genes encoding these enzymes. At least three genes of Δ(1) KSTD (kstD), five genes of KSH subunit A (kshA), and one gene of KSH subunit B of 3-ketosteroid-9α-hydroxylases (kshB) have been found in Mycobacterium sp. VKM Ac-1817D. Strains of Mycobacterium spp. VKM Ac-1815D and 1816D were found to possess at least one kstD, one kshB and two kshA genes. The assembled genome sequence of Mycobacterium sp. VKM Ac-1817D differs from those of 1815D and 1816D strains, whereas these last two are nearly identical, differing by 13 single nucleotide substitutions (SNPs). One of these SNPs is located in the coding region of a kstD gene and corresponds to an amino acid substitution Lys (135) in 1816D for Ser (135) in 1815D. The findings may be useful for targeted genetic engineering of the biocatalysts for biotechnological application. Copyright © 2013. Published by Elsevier Ltd.

  1. Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

    PubMed

    Norman, C; Vidal, S; Palva, E T

    1999-07-01

    Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

  2. Monitoring endogenous enzymes during olive fruit ripening and storage: correlation with virgin olive oil phenolic profiles.

    PubMed

    Hachicha Hbaieb, Rim; Kotti, Faten; García-Rodríguez, Rosa; Gargouri, Mohamed; Sanz, Carlos; Pérez, Ana G

    2015-05-01

    The ability of olive endogenous enzymes β-glucosidase, polyphenol oxidase (PPO) and peroxidase (POX), to determine the phenolic profile of virgin olive oil was investigated. Olives used for oil production were stored for one month at 20 °C and 4 °C and their phenolic content and enzymatic activities were compared to those of ripening olive fruits. Phenolic and volatile profiles of the corresponding oils were also analysed. Oils obtained from fruits stored at 4 °C show similar characteristics to that of freshly harvested fruits. However, the oils obtained from fruits stored at 20 °C presented the lowest phenolic content. Concerning the enzymatic activities, results show that the β-glucosidase enzyme is the key enzyme responsible for the determination of virgin olive oil phenolic profile as the decrease in this enzyme activity after 3 weeks of storage at 20 °C was parallel to a dramatic decrease in the phenolic content of the oils. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Rice Seed Germination Underwater: Morpho-Physiological Responses and the Bases of Differential Expression of Alcoholic Fermentation Enzymes

    PubMed Central

    Miro, Berta; Longkumer, Toshisangba; Entila, Frederickson D.; Kohli, Ajay; Ismail, Abdelbagi M.

    2017-01-01

    The water-, energy-, and labor-intensive system of transplanted puddled rice (Oryza sativa) is steadily being replaced by direct seeding due to the progressive scarcity of these resources. However, the alternate dry direct seeding leads to competition with weeds and poor establishment when soils are flooded. Direct seeded rice capable of anaerobic germination (germination in flooded soil, AG) is ideal, which under rainfed ecosystems would also overcome waterlogging during germination. AG tolerance is associated with faster germination and faster elongation of coleoptiles, with the activities of alcoholic fermentation enzymes replacing aerobic respiration as a source of energy. To better understand the variability in the morpho-physiological responses and in the nature of the alcoholic fermentation enzymes during AG, 21 rice genotypes were studied. The genotypes Khao Hlan On (KHO) and IR42 were used as the tolerant and susceptible checks, respectively. KHO exhibited faster germination, with 82.5% of the coleoptiles emerging out of 10 cm of water within 8 days, whereas IR42 exhibited 20% germination and limited coleoptile growth. Among the test genotypes, four performed well, including two that are drought tolerant. Increased content and activity of the alcoholic fermentation enzymes, alcohol dehydrogenase (ADH1) and acetaldehyde dehydrogenase (ALDH2a and ALDH2b), was noted in KHO under anaerobic than under aerobic conditions and also in comparison with IR42 under AG. Gene transcripts for these enzymes were also more in KHO undergoing AG. However, no major differences were observed between KHO and IR42 in the critical cis-acting regulatory elements, such as the auxin, light, and sugar response elements, in the promoters of ADH1, ALDH2a, and ALDH2b genes. Post-transcriptional and post-translational regulatory mechanisms were implicated for the increased transcript and protein content/activity of the enzymes in KHO by observing four different transcripts of ALDH2a and

  4. Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

    PubMed Central

    Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia

    2014-01-01

    Abstract Significance: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. Recent Advances: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. Critical Issues: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. Future Directions: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world. Antioxid. Redox Signal. 20, 621–639. PMID:23879289

  5. Taste cell-expressed α-glucosidase enzymes contribute to gustatory responses to disaccharides

    PubMed Central

    Sukumaran, Sunil K.; Yee, Karen K.; Iwata, Shusuke; Kotha, Ramana; Quezada-Calvillo, Roberto; Nichols, Buford L.; Mohan, Sankar; Pinto, B. Mario; Shigemura, Noriatsu; Ninomiya, Yuzo; Margolskee, Robert F.

    2016-01-01

    The primary sweet sensor in mammalian taste cells for sugars and noncaloric sweeteners is the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by Tas1r2 and Tas1r3 genes). However, in the absence of T1R2+T1R3 (e.g., in Tas1r3 KO mice), animals still respond to sugars, arguing for the presence of T1R-independent detection mechanism(s). Our previous findings that several glucose transporters (GLUTs), sodium glucose cotransporter 1 (SGLT1), and the ATP-gated K+ (KATP) metabolic sensor are preferentially expressed in the same taste cells with T1R3 provides a potential explanation for the T1R-independent detection of sugars: sweet-responsive taste cells that respond to sugars and sweeteners may contain a T1R-dependent (T1R2+T1R3) sweet-sensing pathway for detecting sugars and noncaloric sweeteners, as well as a T1R-independent (GLUTs, SGLT1, KATP) pathway for detecting monosaccharides. However, the T1R-independent pathway would not explain responses to disaccharide and oligomeric sugars, such as sucrose, maltose, and maltotriose, which are not substrates for GLUTs or SGLT1. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we found that taste cells express multiple α-glycosidases (e.g., amylase and neutral α glucosidase C) and so-called intestinal “brush border” disaccharide-hydrolyzing enzymes (e.g., maltase-glucoamylase and sucrase-isomaltase). Treating the tongue with inhibitors of disaccharidases specifically decreased gustatory nerve responses to disaccharides, but not to monosaccharides or noncaloric sweeteners, indicating that lingual disaccharidases are functional. These taste cell-expressed enzymes may locally break down dietary disaccharides and starch hydrolysis products into monosaccharides that could serve as substrates for the T1R-independent sugar sensing pathways. PMID:27162343

  6. Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection

    NASA Astrophysics Data System (ADS)

    Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

    2016-03-01

    Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most

  7. Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

    PubMed

    Luo, Ling; Meng, Han; Gu, Ji-Dong

    2017-07-15

    Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. SGD1, a key enzyme in tocopherol biosynthesis, is essential for plant development and cold tolerance in rice.

    PubMed

    Wang, Di; Wang, Yunlong; Long, Wuhua; Niu, Mei; Zhao, Zhigang; Teng, Xuan; Zhu, Xiaopin; Zhu, Jianping; Hao, Yuanyuan; Wang, Yongfei; Liu, Yi; Jiang, Ling; Wang, Yihua; Wan, Jianmin

    2017-07-01

    Tocopherols, a group of Vitamin E compounds, are essential components of the human diet. In contrast to well documented roles in animals, the functions of tocopherols in plants are less understood. In this study, we characterized two allelic rice dwarf mutant lines designated sgd1-1 and sgd1-2 (small grain and dwarf1). Histological observations showed that the dwarf phenotypes were mainly due to cell elongation defects. A map-based cloning strategy and subsequent complementation test showed that SGD1 encodes homogentisate phytyltransferase (HPT), a key enzyme in tocopherol biosynthesis. Mutation of SGD1 resulted in tocopherol deficiency in both sgd1mutants. No oxidant damage was detected in the sgd1 mutants. Further analysis showed that sgd1-2 was hypersensitive to cold stress. Our results indicate that SGD1 is essential for plant development and cold tolerance in rice. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Overexpression of gene encoding the key enzyme involved in proline-biosynthesis (PuP5CS) to improve salt tolerance in switchgrass (Panicum virgatum L.).

    PubMed

    Guan, Cong; Huang, Yan-Hua; Cui, Xin; Liu, Si-Jia; Zhou, Yun-Zhuan; Zhang, Yun-Wei

    2018-05-25

    Genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant salt stress tolerance. Switchgrass (Panicum virgatum L.) has developed into a dedicated bioenergy crop. To improve the biomass production of switchgrass grown on different types of soil, abiotic stress tolerance traits are considered for its genetic improvement. Proline accumulation is a widespread response when plants are subjected to abiotic stresses such as drought, cold and salinity. In plants, P5CS gene encodes the key regulatory enzyme that plays a crucial role in proline biosynthesis. Here, we introduced the PuP5CS gene (from Puccinellia chinampoensis) into switchgrass by Agrobacterium-mediated transformation. Transgenic lines overexpressing the PuP5CS gene showed phenotypic advantages, in leaf width, internode diameter, internode length, tiller numbers and precocious flowering under normal conditions, and the transgenic lines displayed better regenerative capacity in forming more tillers after harvest. Moreover, the PuP5CS gene enhanced the salt tolerance of transgenic switchgrass by altering a wide range of physiological responses. In accordance with the physiological results, histological analysis of cross sections through the leaf blade showed that the areas of bulliform cells and bundle sheath cells were significantly increased in PuP5CS-overexpressing leaves. The expression levels of ROS scavenging-associated genes in transgenic plants were higher than in control plants under salt stress. The results show that genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant stress tolerance.

  10. The Classification and Evolution of Enzyme Function

    PubMed Central

    Martínez Cuesta, Sergio; Rahman, Syed Asad; Furnham, Nicholas; Thornton, Janet M.

    2015-01-01

    Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes. PMID:25986631

  11. Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis)

    PubMed Central

    Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

    2015-01-01

    Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants’ growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., ‘Photosynthesis’), GO terms (e.g., ‘response to karrikin’) and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology. PMID:25901577

  12. Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

    PubMed Central

    Pallottini, Valentina; Canuti, Lorena; De Canio, Michele; Urbani, Andrea; Marzano, Valeria; Cornetta, Tommaso; Stano, Pasquale; Giovanetti, Anna; Stella, Lorenzo; Canini, Antonella; Federici, Giorgio; Ricci, Giorgio

    2010-01-01

    Background In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions. Methodology/Principal Findings Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization. Conclusions/Significance The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and

  13. Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots

    PubMed Central

    Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

    2013-01-01

    Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g−1 of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l−1 EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. PMID:23170978

  14. Mercury-induced oxidative stress and impact on antioxidant enzymes in Chlamydomonas reinhardtii.

    PubMed

    Elbaz, Abdelrahman; Wei, Yuan Yuan; Meng, Qian; Zheng, Qi; Yang, Zhi Min

    2010-10-01

    Investigation of mercury toxicology in green algae is of great importance from ecological point of view, because mercury has become a major contaminant in recent years. In higher plants, accumulation of mercury modifies many aspects of cellular functions. However, the process that mercury exerts detrimental effects on green algae is largely unknown. In this study, we performed an experiment focusing on the biological responses of Chlamydomonas reinhardtii, a unicellular model organism, to Hg(2+)-induced toxicity. C. reinhardtii was exposed to 0, 1, 2, 4, 6, and 8 μM Hg in media. Concentrations of Hg were negatively correlated with the cell growth. Treatment with Hg induced accumulation of reactive oxygen species and peroxidative products. Endogenous proline levels increased in Hg-exposed algae. Hg exposure activated superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). To get insights into the molecular response, a RT-PCR-based assay was performed to analyze the transcript abundance of Mn-SOD, CAT and APX. Our analysis revealed that expression of the genes was up-regulated by Hg exposure, with a pattern similar to the enzyme activities. Additional investigation was undertaken on the effect of Hg on the transcript amount of ∆(1)-pyrroline-5-carboxylate synthetase, a key enzyme of proline biosynthesis and on that of heme oxygenase-1 (HO-1), an enzyme regulating heavy metal tolerance. Expressions of both P5CS and HO-1 were up-regulated by Hg. These data indicate that Hg-induced oxidative stress was responsible for the disturbance of the growth and antioxidant defensive systems in C. reinhardtii.

  15. Key ecological responses to nitrogen are altered by climate change

    USGS Publications Warehouse

    Greaver, T.L.; Clark, C.M.; Compton, J.E.; Vallano, D.; Talhelm, A. F.; Weaver, C.P.; Band, L.E.; Baron, Jill S.; Davidson, E.A.; Tague, C.L.; Felker-Quinn, E.; Lynch, J.A.; Herrick, J.D.; Liu, L.; Goodale, C.L.; Novak, K. J.; Haeuber, R. A.

    2016-01-01

    Climate change and anthropogenic nitrogen deposition are both important ecological threats. Evaluating their cumulative effects provides a more holistic view of ecosystem vulnerability to human activities, which would better inform policy decisions aimed to protect the sustainability of ecosystems. Our knowledge of the cumulative effects of these stressors is growing, but we lack an integrated understanding. In this Review, we describe how climate change alters key processes in terrestrial and freshwater ecosystems related to nitrogen cycling and availability, and the response of ecosystems to nitrogen addition in terms of carbon cycling, acidification and biodiversity.

  16. Key ecological responses to nitrogen are altered by climate change

    NASA Astrophysics Data System (ADS)

    Greaver, T. L.; Clark, C. M.; Compton, J. E.; Vallano, D.; Talhelm, A. F.; Weaver, C. P.; Band, L. E.; Baron, J. S.; Davidson, E. A.; Tague, C. L.; Felker-Quinn, E.; Lynch, J. A.; Herrick, J. D.; Liu, L.; Goodale, C. L.; Novak, K. J.; Haeuber, R. A.

    2016-09-01

    Climate change and anthropogenic nitrogen deposition are both important ecological threats. Evaluating their cumulative effects provides a more holistic view of ecosystem vulnerability to human activities, which would better inform policy decisions aimed to protect the sustainability of ecosystems. Our knowledge of the cumulative effects of these stressors is growing, but we lack an integrated understanding. In this Review, we describe how climate change alters key processes in terrestrial and freshwater ecosystems related to nitrogen cycling and availability, and the response of ecosystems to nitrogen addition in terms of carbon cycling, acidification and biodiversity.

  17. Dose response of bone-targeted enzyme replacement for murine hypophosphatasia.

    PubMed

    Yadav, Manisha C; Lemire, Isabelle; Leonard, Pierre; Boileau, Guy; Blond, Laurent; Beliveau, Martin; Cory, Esther; Sah, Robert L; Whyte, Michael P; Crine, Philippe; Millán, José Luis

    2011-08-01

    Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2mg/kg for 43days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED(80) (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PP(i) concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP in patients, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP to mice. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Dose response of bone-targeted enzyme replacement for murine hypophosphatasia

    PubMed Central

    Yadav, Manisha C.; Lemire, Isabelle; Leonard, Pierre; Boileau, Guy; Blond, Laurent; Beliveau, Martin; Cory, Esther; Sah, Robert L.; Whyte, Michael P.; Crine, Philippe; Millán, José Luis

    2011-01-01

    Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD10, renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2−/−) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2−/− mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2 mg/kg for 43 days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED80 (the dose prevents the bone defects in 80% of mice) was 3.2, 2.8 and 2.9 mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PPi concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials. PMID:21458605

  19. The Action of Antidiabetic Plants of the Canadian James Bay Cree Traditional Pharmacopeia on Key Enzymes of Hepatic Glucose Homeostasis

    PubMed Central

    Nachar, Abir; Vallerand, Diane; Musallam, Lina; Lavoie, Louis; Arnason, John; Haddad, Pierre S.

    2013-01-01

    We determined the capacity of putative antidiabetic plants used by the Eastern James Bay Cree (Canada) to modulate key enzymes of gluconeogenesis and glycogen synthesis and key regulating kinases. Glucose-6-phosphatase (G6Pase) and glycogen synthase (GS) activities were assessed in cultured hepatocytes treated with crude extracts of seventeen plant species. Phosphorylation of AMP-dependent protein kinase (AMPK), Akt, and Glycogen synthase kinase-3 (GSK-3) were probed by Western blot. Seven of the seventeen plant extracts significantly decreased G6Pase activity, Abies balsamea and Picea glauca, exerting an effect similar to insulin. This action involved both Akt and AMPK phosphorylation. On the other hand, several plant extracts activated GS, Larix laricina and A. balsamea, far exceeding the action of insulin. We also found a significant correlation between GS stimulation and GSK-3 phosphorylation induced by plant extract treatments. In summary, three Cree plants stand out for marked effects on hepatic glucose homeostasis. P. glauca affects glucose production whereas L. laricina rather acts on glucose storage. However, A. balsamea has the most promising profile, simultaneously and powerfully reducing G6Pase and stimulating GS. Our studies thus confirm that the reduction of hepatic glucose production likely contributes to the therapeutic potential of several antidiabetic Cree traditional medicines. PMID:23864882

  20. Entropy driven key-lock assembly.

    PubMed

    Odriozola, G; Jiménez-Angeles, F; Lozada-Cassou, M

    2008-09-21

    The effective interaction between a sphere with an open cavity (lock) and a spherical macroparticle (key), both immersed in a hard sphere fluid, is studied by means of Monte Carlo simulations. As a result, a two-dimensional map of the key-lock effective interaction potential is constructed, which leads to the proposal of a self-assembling mechanism: There exists trajectories through which the key-lock pair could assemble avoiding trespassing potential barriers. Hence, solely the entropic contribution can induce their self-assembling even in the absence of attractive forces. This study points out the solvent contribution within the underlying mechanisms of substrate-protein assemblydisassembly processes, which are important steps of the enzyme catalysis and protein mediated transport.

  1. Entropy driven key-lock assembly

    NASA Astrophysics Data System (ADS)

    Odriozola, G.; Jiménez-Ángeles, F.; Lozada-Cassou, M.

    2008-09-01

    The effective interaction between a sphere with an open cavity (lock) and a spherical macroparticle (key), both immersed in a hard sphere fluid, is studied by means of Monte Carlo simulations. As a result, a two-dimensional map of the key-lock effective interaction potential is constructed, which leads to the proposal of a self-assembling mechanism: There exists trajectories through which the key-lock pair could assemble avoiding trespassing potential barriers. Hence, solely the entropic contribution can induce their self-assembling even in the absence of attractive forces. This study points out the solvent contribution within the underlying mechanisms of substrate-protein assembly/disassembly processes, which are important steps of the enzyme catalysis and protein mediated transport.

  2. Geometric and electronic structure contributions to function in non-heme iron enzymes.

    PubMed

    Solomon, Edward I; Light, Kenneth M; Liu, Lei V; Srnec, Martin; Wong, Shaun D

    2013-11-19

    Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)═O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for

  3. The Classification and Evolution of Enzyme Function.

    PubMed

    Martínez Cuesta, Sergio; Rahman, Syed Asad; Furnham, Nicholas; Thornton, Janet M

    2015-09-15

    Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Dioxygen Binding, Activation, and Reduction to H2O by Cu Enzymes.

    PubMed

    Solomon, Edward I

    2016-07-05

    Oxygen intermediates in copper enzymes exhibit unique spectroscopic features that reflect novel geometric and electronic structures that are key to reactivity. This perspective will describe: (1) the bonding origin of the unique spectroscopic features of the coupled binuclear copper enzymes and how this overcomes the spin forbiddenness of O2 binding and activates monooxygenase activity, (2) how the difference in exchange coupling in the non-coupled binuclear Cu enzymes controls the reaction mechanism, and (3) how the trinuclear Cu cluster present in the multicopper oxidases leads to a major structure/function difference in enabling the irreversible reductive cleavage of the O-O bond with little overpotential and generating a fully oxidized intermediate, different from the resting enzyme studied by crystallography, that is key in enabling fast PCET in the reductive half of the catalytic cycle.

  5. Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

    PubMed

    Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

    2016-03-21

    Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

  6. Suppression of 9-cis-Epoxycarotenoid Dioxygenase, Which Encodes a Key Enzyme in Abscisic Acid Biosynthesis, Alters Fruit Texture in Transgenic Tomato1[W][OA

    PubMed Central

    Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

    2012-01-01

    Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp). PMID:22108525

  7. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  8. Production of Enzymes from Marine Actinobacteria.

    PubMed

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

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

  9. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maltz, Lauren

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

  10. Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT

    PubMed Central

    Rempel, Brian P.; Price, Eric W.

    2017-01-01

    Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents. PMID:28927325

  11. Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT.

    PubMed

    Rempel, Brian P; Price, Eric W; Phenix, Christopher P

    2017-01-01

    Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

  12. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    NASA Astrophysics Data System (ADS)

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

  13. Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots.

    PubMed

    Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

    2013-05-01

    Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g⁻¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l⁻¹ EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  14. Expression and Enzyme Activity of Catalase in Chilo suppressalis (Lepidoptera: Crambidae) Is Responsive to Environmental Stresses.

    PubMed

    Lu, Yanhui; Bai, Qi; Zheng, Xusong; Lu, Zhongxian

    2017-08-01

    Catalase (CAT) is an important antioxidant enzyme that protects organisms against oxidative stresses by eliminating hydrogen peroxide. In this study, we cloned and characterized a full-length cDNA of CAT from Chilo suppressalis (CsCAT) and examined the influence of environmental stresses on CsCAT expression and enzyme activity. The cDNA contains a 1659-bp open reading frame encoding a polypeptide of 553 amino acids most closely related (90.14%) to Papilio polytes catalases. The CsCAT was expressed in all developmental stages with the highest expression in the fat body, and the CsCAT enzyme activity closely mirrored its observed mRNA expression patterns. The CsCAT mRNA was up-regulated when the larvae were exposed to high temperature (≥30 °C), insecticides (abamectin and chlorantraniliprole), chemicals (H2O2, CHP, CdCl2, and CuSO4), and a dead-end trap plant (vetiver grass), and the CsCAT enzyme activity again mirrored the observed CsCAT expression patterns. These results suggest that up-regulation of CsCAT may enhance the defense response of C. suppressalis by weakening the effects of environmental stresses, and provide insight into the role of CsCAT during development of C. suppressalis. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. UHPLC-QqQ-MS/MS identification, quantification of polyphenols from Passiflora subpeltata fruit pulp and determination of nutritional, antioxidant, α-amylase and α-glucosidase key enzymes inhibition properties.

    PubMed

    Shanmugam, Saravanan; Gomes, Isla Alcântara; Denadai, Marina; Dos Santos Lima, Bruno; de Souza Araújo, Adriano Antunes; Narain, Narendra; Neta, Maria Terezinha Santos Leite; Serafini, Mairim Russo; Quintans-Júnior, Lucindo José; Thangaraj, Parimelazhagan

    2018-06-01

    The diabetic key enzymes inhibition, nutritional, antioxidant activity and bioactive compounds identification of Passiflora subpeltata fruit pulp were investigated. Fifteen polyphenolic compounds including protocatechuic acid, ferulic acid, vanillic acid, epicatechin, p-coumaric acid, cinnamic acid, eriodictyol and quercetin-3-glucoside were identified in the pulp of this species by using UHPLC-QqQ-MS/MS analysis. The total carbohydrates and crude protein contents in fruit pulp were 2.62 mg glucose equivalent/g sample fruit pulp and 8.80 mg BSA equivalent/g sample fruit pulp, respectively. The fresh fruit pulp of P. subpeltata contained high total phenolic (724.76 mg GAE/g sample) content and it revealed very high DPPH • (IC 50 of 5.667 μg/mL) and ABTS +• (6794.96 μM trolox equivalent/g sample) scavenging activities. In the key enzymes assays useful for diabetic inhibition the fresh fruit pulp characterized maximum inhibition of α-amylase and α-glucosidase IC 50 of 18.69 and 32.63 μg/mL, respectively. Thus, these results lead to conclude that this fruit specie could be very useful source in nutraceutical products preparations for Type 2 diabetic suffering humans. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. A new amperometric enzyme electrode for alcohol determination.

    PubMed

    Gülce, H; Gülce, A; Kavanoz, M; Coşkun, H; Yildiz, A

    2002-06-01

    A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.

  17. Toxicogenomics and cancer risk assessment: a framework for key event analysis and dose-response assessment for nongenotoxic carcinogens.

    PubMed

    Bercu, Joel P; Jolly, Robert A; Flagella, Kelly M; Baker, Thomas K; Romero, Pedro; Stevens, James L

    2010-12-01

    In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

    NASA Technical Reports Server (NTRS)

    Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

    1994-01-01

    Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

  19. -Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression.

    PubMed

    Mattice, Amanda M S; MacLean, Isabelle A; Childers, Christine L; Storey, Kenneth B

    2018-01-01

    Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles ( Trachemys scripta elegans ). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I 50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I 50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.

  20. Genetic polymorphisms in 5-Fluorouracil-related enzymes predict pathologic response after neoadjuvant chemoradiation for rectal cancer.

    PubMed

    Nelson, Bailey; Carter, Jane V; Eichenberger, Maurice R; Netz, Uri; Galandiuk, Susan

    2016-11-01

    Many patients with rectal cancer undergo preoperative neoadjuvant chemoradiation, with approximately 70% exhibiting pathologic downstaging in response to treatment. Currently, there is no accurate test to predict patients who are likely to be complete responders to therapy. 5-Fluorouracil is used regularly in the neoadjuvant treatment of rectal cancer. Genetic polymorphisms affect the activity of thymidylate synthase, an enzyme involved in 5-Fluorouracil metabolism, which may account for observed differences in response to neoadjuvant treatment between patients. Detection of genetic polymorphisms might identify patients who are likely to have a complete response to neoadjuvant therapy and perhaps allow them to avoid operation. DNA was isolated from whole blood taken from patients with newly diagnosed rectal cancer who received neoadjuvant therapy (n = 50). Response to therapy was calculated with a tumor regression score based on histology from the time of operation. Polymerase chain reaction was performed targeting the promoter region of thymidylate synthase. Polymerase chain reaction products were separated using electrophoresis to determine whether patients were homozygous for a double-tandem repeat (2R), a triple-tandem repeat (3R), or were heterozygous (2R/3R). A single nucleotide polymorphism, 3G or 3C, also may be present in the second repeat unit of the triple-tandem repeat allele. Restriction fragment length polymorphism assays were performed in patients with at least one 3R allele using HaeIII. Patients with at least 1 thymidylate synthase 3G allele were more likely to have a complete or partial pathologic response to 5-Fluorouracil neoadjuvant therapy (odds ratio 10.4; 95% confidence interval, 1.3-81.6; P = .01) than those without at least one 3G allele. Identification of rectal cancer patients with specific genetic polymorphisms in enzymes involved in 5-Fluorouracil metabolism seems to predict the likelihood of complete or partial pathologic response

  1. Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide

    PubMed Central

    2018-01-01

    Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment. PMID:29570264

  2. Strategic enzyme patterning for microfluidic biofuel cells

    NASA Astrophysics Data System (ADS)

    Kjeang, E.; Sinton, D.; Harrington, D. A.

    The specific character of biological enzyme catalysts enables combined fuel and oxidant channels and simplified non-compartmentalized fuel cell assemblies. In this work, a microstructured enzymatic biofuel cell architecture is proposed, and species transport phenomena combined with consecutive chemical reactions are studied computationally in order to provide guidelines for optimization. This is the first computational study of this technology, and a 2D CFD model for species transport coupled with laminar fluid flow and Michaelis-Menten enzyme kinetics is established. It is shown that the system is reaction rate limited, indicating that enzyme specific turnover numbers are key parameters for biofuel cell performance. Separated and mixed enzyme patterns in different proportions are analyzed for various Peclet numbers. High fuel utilization is achieved in the diffusion dominated and mixed species transport regimes with separated enzymes arranged in relation to individual turnover rates. However, the Peclet number has to be above a certain threshold value to obtain satisfying current densities. The mixed transport regime is particularly attractive while current densities are maintained close to maximum levels. Optimum performance is achieved by mixed enzyme patterning tailored with respect to individual turnover rates, enabling high current densities combined with nearly complete fuel utilization.

  3. Developing Master Keys to Brain Pathology, Cancer and Aging from the Structural Biology of Proteins Controlling Reactive Oxygen Species and DNA Repair

    PubMed Central

    Perry, J. Jefferson P.; Fan, Li; Tainer, John A.

    2007-01-01

    This review is focused on proteins with key roles in pathways controlling either reactive oxygen species or DNA damage responses, both of which are essential for preserving the nervous system. An imbalance of reactive oxygen species or inappropriate DNA damage response likely causes mutational or cytotoxic outcomes, which may lead to cancer and/or aging phenotypes. Moreover, individuals with hereditary disorders in proteins of these cellular pathways have significant neurological abnormalities. Mutations in a superoxide dismutase, which removes oxygen free radicals, may cause the neurodegenerative disease amyotrophic lateral sclerosis. Additionally, DNA repair disorders that affect the brain to varying extents include ataxia-telangiectasia-like disorder, Cockayne syndrome or Werner syndrome. Here, we highlight recent advances gained through structural biochemistry studies on enzymes linked to these disorders and other related enzymes acting within the same cellular pathways. We describe the current understanding of how these vital proteins coordinate chemical steps and integrate cellular signaling and response events. Significantly, these structural studies may provide a set of master keys to developing a unified understanding of the survival mechanisms utilized after insults by reactive oxygen species and genotoxic agents, and also provide a basis for developing an informed intervention in brain tumor and neurodegenerative disease progression. PMID:17174478

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

    PubMed

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

    2017-11-01

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

  5. Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

    PubMed Central

    Jimeno, P; Garcia-Perez, A I; Luque, J; Pinilla, M

    1991-01-01

    Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes. PMID:1656939

  6. Evidence for a regulatory loop between cholecystokinin (CCK) and tryptic enzyme activity in Atlantic cod larvae (Gadus morhua).

    PubMed

    Tillner, Robert; Rønnestad, Ivar; Harboe, Torstein; Ueberschär, Bernd

    2013-11-01

    In order to maximize protein digestion, the release of enzymes into the gut lumen is closely controlled by a regulatory loop. Cholecystokinin (CCK) is among the enteric hormones that play a key role in the control of digestive enzyme secretion, but its role in first-feeding larvae is still unclear and may differ between species. However, in all marine fish larvae that have not developed a stomach by first-feeding, trypsin is the most important proteolytic enzyme. In order to examine the regulation and feedback mechanisms in the gut of larval cod, we therefore studied the interactions between cholecystokinin and tryptic enzyme activity following the administration of solutions containing test substances directly into the gut. We tube-fed a single dose of physiological saline solution containing either CCK, CCK antagonist, trypsin inhibitor, phytohemagglutinin (PHA; a possible trigger for the digestive response) or physiological saline alone, while a further control group was left untreated. We then followed the response in CCK and tryptic enzyme activity for 0.5-8h after the administration. We performed the experiment on larvae at 26day post first-feeding, which is before the stomach has evolved and the size of the larvae allows easier handling. Individual larvae were analyzed for CCK and tryptic enzyme activity using radioimmunoassay and fluorimetric techniques respectively. Both factors varied over time in the untreated control group, possibly due to an endogenous daily rhythm. The higher CCK levels at 4h and 8h in the saline-injected group may be caused by reflexes initiated by distension of the gut. An increase in tryptic enzyme activity after injection of CCK supports the hypothesis that this hormone plays a part in the release of pancreatic enzymes in larval cod at this developmental stage. However, administration of a CCK antagonist and a trypsin inhibitor did not reveal conclusive results, probably due to the relatively low concentrations used. The response

  7. Enzyme-triggered compound release using functionalized antimicrobial peptide derivatives† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04435b Click here for additional data file.

    PubMed Central

    Kashibe, Masayoshi; Matsumoto, Kengo; Hori, Yuichiro

    2017-01-01

    Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool. PMID:28451373

  8. Inhibition of key enzymes linked to type 2 diabetes by compounds isolated from Aframomum melegueta fruit.

    PubMed

    Mohammed, Aminu; Gbonjubola, Victoria Awolola; Koorbanally, Neil Anthony; Islam, Md Shahidul

    2017-12-01

    The use of Aframomum melegueta K. Schum. (Zingiberaceae) fruit for treatment of diabetes has recently been established in Nigeria. However, compounds responsible for the antidiabetic action have not been identified. The present study carried out the bioassay-guided isolation of possible bioactive compounds responsible for the antidiabetic action of A. melegueta fruit. The A. melegueta fruit was sequentially extracted using ethyl acetate (EtOAc), ethanol and water, and the most active extract (EtOAc) was subjected to column chromatography on a silica gel column using solvent gradient systems of hexane (HEX):EtOAc and EtOAc:MeOH and the isolation of compounds was guided by α-glycosidase and α-amylase inhibitory activities at various concentrations (30-240 μg/mL). According to the results, 3 arylalkanes, 6-paradol (1), 6-shogaol (2) and 6-gingerol (3) and a pentacyclic triterpene, oleanolic acid (4) were isolated from A. melegueta fruit. All the compounds exhibited inhibitory effects against α-amylase and α-glucosidase. 6-Gingerol (3) and oleanolic acid (4) showed higher inhibitory activity against α-amylase (IC 50 : 6-gingerol: 81.78 ± 7.79 μM; oleanolic acid: 91.72 ± 1.63 μM) and α-glucosidase (IC 50 : 6-gingerol: 21.55 ± 0.45 μM; oleanolic acid: 17.35 ± 0.88 μM) compared to the standard drug, acarbose and other isolated compounds. The kinetics of the enzyme action of the compounds showed a noncompetitive mode of inhibition. The data of this study suggest that the 6-gingerol (3) and oleanolic acid (4) showed higher α-amylase and α-glucosidase inhibitory action and therefore could be responsible for the antidiabetic activity of A. melegueta fruit.

  9. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

    PubMed

    Vieille, C; Zeikus, G J

    2001-03-01

    Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

  10. Inhibitory effect of leaves extracts of Ocimum basilicum and Ocimum gratissimum on two key enzymes involved in obesity and hypertension in vitro

    PubMed Central

    Irondi, Emmanuel Anyachukwu; Agboola, Samson Olalekan; Oboh, Ganiyu; Boligon, Aline Augusti

    2016-01-01

    Aim: To evaluate the phenolics composition and inhibitory effect of the leaves extracts of Ocimum basilicum and Ocimum gratissimum on two key enzymes (pancreatic lipase [PL] and angiotensin 1-converting enzyme [ACE]) involved in obesity and hypertension in vitro. Materials and Methods: The phenolics (flavonoids and phenolic acids) were quantified using high-performance liquid chromatography coupled with diode array detection. PL and ACE inhibitory effects; DPPH* and ABTS*+ scavenging activities of the extracts were tested using spectrophotometric methods. Results: O. basilicum had the following major phenolics: Rutin, quercetin, and quercitrin (flavonoids); caffeic, chlorogenic, and gallic acids (phenolic acids); while O. gratissimum had the following major phenolics: Rutin, quercitrin, and luteolin (flavonoids); ellagic and chlorogenic acids (phenolic acids). “Extracts of both plants inhibited PL and ACE; scavenged DPPH* in a dose-dependent manner”. O. gratissimum extract was more potent in inhibiting PL (IC50: 20.69 µg/mL) and ACE (IC50: 29.44 µg/mL) than O. basilicum (IC50: 52.14 µg/mL and IC50: 64.99 µg/mL, against PL and ACE, respectively). O. gratissimum also scavenged DPPH* and ABTS*+ more than O. basilicum. Conclusion: O. basilicum and O. gratissimum leaves could be used as functional foods for the management of obesity and obesity-related hypertension. However, O. gratissimum may be more effective than O. basilicum. PMID:27757270

  11. Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes

    PubMed Central

    Baker, Perrin; Seah, Stephen Y. K.

    2012-01-01

    Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed. PMID:24688644

  12. Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

    PubMed

    Badur, Ahmet H; Plutz, Matthew J; Yalamanchili, Geethika; Jagtap, Sujit Sadashiv; Schweder, Thomas; Unfried, Frank; Markert, Stephanie; Polz, Martin F; Hehemann, Jan-Hendrik; Rao, Christopher V

    2017-08-04

    Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Fermentation, Respiration & Enzyme Specificity: A Simple Device & Key Experiments with Yeast.

    ERIC Educational Resources Information Center

    Reinking, Larry N.; And Others

    1994-01-01

    Using graphs and diagrams, the authors describe a simple fermentation chamber and provide key experiments that can be used in the classroom to give students meaningful insight into metabolic processes. (ZWH)

  14. Protein Surface Softness Is the Origin of Enzyme Cold-Adaptation of Trypsin

    PubMed Central

    Isaksen, Geir Villy; Åqvist, Johan; Brandsdal, Bjørn Olav

    2014-01-01

    Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution. PMID:25165981

  15. Enzyme immobilisation in biocatalysis: why, what and how.

    PubMed

    Sheldon, Roger A; van Pelt, Sander

    2013-08-07

    In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

  16. Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants - A review.

    PubMed

    Bilal, Muhammad; Asgher, Muhammad; Parra-Saldivar, Roberto; Hu, Hongbo; Wang, Wei; Zhang, Xuehong; Iqbal, Hafiz M N

    2017-01-15

    In the twenty-first century, chemical and associated industries quest a transition prototype from traditional chemical-based concepts to a greener, sustainable and environmentally-friendlier catalytic alternative, both at the laboratory and industrial scale. In this context, bio-based catalysis offers numerous benefits along with potential biotechnological and environmental applications. The bio-based catalytic processes are energy efficient than conventional methodologies under moderate processing, generating no and negligible secondary waste pollution. Thanks to key scientific advances, now, solid-phase biocatalysts can be economically tailored on a large scale. Nevertheless, it is mandatory to recover and reprocess the enzyme for their commercial feasibility, and immobilization engineering can efficiently accomplish this challenge. The first part of the present review work briefly outlines the immobilization of lignin-modifying enzymes (LMEs) including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase of white-rot fungi (WRF). Whereas, in the second part, a particular emphasis has been given on the recent achievements of carrier-immobilized LMEs for the degradation, decolorization, or detoxification of industrial dyes and dye-based industrial wastewater effluents. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. The Potential Coordination of the Heat-Shock Proteins and Antioxidant Enzyme Genes of Aphidius gifuensis in Response to Thermal Stress

    PubMed Central

    Kang, Zhi-Wei; Liu, Fang-Hua; Liu, Xiang; Yu, Wen-Bo; Tan, Xiao-Ling; Zhang, Shi-Ze; Tian, Hong-Gang; Liu, Tong-Xian

    2017-01-01

    Aphidius gifuensis is one of the most important aphid natural enemies and has been successfully used to control Myzys persicae and other aphid species. High temperature in summer is one of the key barriers for the application of A. gifuensis in the field and greenhouse. In this work, we investigated the biological performance of A. gifuensis and the response of heat-shock proteins and antioxidant enzymes under high temperature. The results showed that A. gifuensis could not survive at 40°C and female exhibited a higher survival in 35°C. Furthermore, the short term exposure to high temperature negatively affected the performance of A. gifuensis especially parasitism efficiency. Under short-term heating, the expression of AgifsHSP, Agifl(2)efl, AgifHSP70, AgifHSP70-4 and AgifHSP90 showed an increased trend, whereas AgifHSP10 initially increased and then decreased. In 35°C, the expressions of Agifl(2)efl, AgifHSP70-4 and AgifHSP90 in female were higher than those in male, whereas the expression of AgifHSP70 exhibited an opposite trend. Besides the HSPs, we also quantified the expression levels of 11 antioxidant enzyme genes: AgifPOD, AgifSOD1, AgifSOD2, AgifSOD3, AgifCAT1, AgifCAT2, AgifGST1, AgifGST2, AgifGST3, AgifGST4 and AgifGST5. We found that the sex-specific expression of AgifSOD2, AgifSOD3, AgifPOD, AgifGST1 and AgifGST3 were highly consistent with sex-specific heat shock survival rates at 35°C. Furthermore, when the temperature was above 30°C, the activities of GST, SOD, CAT and POD were significantly increased; however, there was no significant difference of the CAT activity between the male and female at 35°C. Collectively, all of these results suggested that the protection of thermal damage is coordinated by HSPs and antioxidant enzymes in A. gifuensis. Based on the heat tolerance abilities of many aphid natural enemies, we also discussed an integrated application strategy of many aphid enemies in summer. PMID:29234290

  18. Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins.

    PubMed

    Balsano, Evelyn; Esterhuizen-Londt, Maranda; Hoque, Enamul; Lima, Stephan Pflugmacher

    2017-08-01

    To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l -1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l -1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

  19. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach.

    PubMed

    Glorieux, Christophe; Calderon, Pedro Buc

    2017-09-26

    This review is centered on the antioxidant enzyme catalase and will present different aspects of this particular protein. Among them: historical discovery, biological functions, types of catalases and recent data with regard to molecular mechanisms regulating its expression. The main goal is to understand the biological consequences of chronic exposure of cells to hydrogen peroxide leading to cellular adaptation. Such issues are of the utmost importance with potential therapeutic extrapolation for various pathologies. Catalase is a key enzyme in the metabolism of H2O2 and reactive nitrogen species, and its expression and localization is markedly altered in tumors. The molecular mechanisms regulating the expression of catalase, the oldest known and first discovered antioxidant enzyme, are not completely elucidated. As cancer cells are characterized by an increased production of reactive oxygen species (ROS) and a rather altered expression of antioxidant enzymes, these characteristics represent an advantage in terms of cell proliferation. Meanwhile, they render cancer cells particularly sensitive to an oxidant insult. In this context, targeting the redox status of cancer cells by modulating catalase expression is emerging as a novel approach to potentiate chemotherapy.

  20. The position of a key tyrosine in dTDP-4-Keto-6-deoxy-D-glucose-5-epimerase (EvaD) alters the substrate profile for this RmlC-like enzyme.

    PubMed

    Merkel, Alexandra B; Major, Louise L; Errey, James C; Burkart, Michael D; Field, Robert A; Walsh, Christopher T; Naismith, James H

    2004-07-30

    Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 A structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

  1. Carbonic anhydrase enzymes regulate mast cell–mediated inflammation

    PubMed Central

    Soteropoulos, Patricia

    2016-01-01

    Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine–mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2–associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy–like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell–mediated inflammation. PMID:27526715

  2. Differential expression of glucose-metabolizing enzymes in multiple sclerosis lesions.

    PubMed

    Nijland, Philip G; Molenaar, Remco J; van der Pol, Susanne M A; van der Valk, Paul; van Noorden, Cornelis J F; de Vries, Helga E; van Horssen, Jack

    2015-12-04

    Demyelinated axons in multiple sclerosis (MS) lesions have an increased energy demand in order to maintain conduction. However, oxidative stress-induced mitochondrial dysfunction likely alters glucose metabolism and consequently impairs neuronal function in MS. Imaging and pathological studies indicate that glucose metabolism is altered in MS, although the underlying mechanisms and its role in neurodegeneration remain elusive. We investigated expression patterns of key enzymes involved in glycolysis, tricarboxylic acid (TCA) cycle and lactate metabolism in well-characterized MS tissue to establish which regulators of glucose metabolism are involved in MS and to identify underlying mechanisms. Expression levels of glycolytic enzymes were increased in active and inactive MS lesions, whereas expression levels of enzymes involved in the TCA cycle were upregulated in active MS lesions, but not in inactive MS lesions. We observed reduced expression and production capacity of mitochondrial α-ketoglutarate dehydrogenase (αKGDH) in demyelinated axons, which correlated with signs of axonal dysfunction. In inactive lesions, increased expression of lactate-producing enzymes was observed in astrocytes, whereas lactate-catabolising enzymes were mainly detected in axons. Our results demonstrate that the expression of various enzymes involved in glucose metabolism is increased in both astrocytes and axons in active MS lesions. In inactive MS lesions, we provide evidence that astrocytes undergo a glycolytic shift resulting in enhanced astrocyte-axon lactate shuttling, which may be pivotal for the survival of demyelinated axons. In conclusion, we show that key enzymes involved in energy metabolism are differentially expressed in active and inactive MS lesions. Our findings imply that, in addition to reduced oxidative phosphorylation activity, other bioenergetic pathways are affected as well, which may contribute to ongoing axonal degeneration in MS.

  3. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

    PubMed Central

    Vieille, Claire; Zeikus, Gregory J.

    2001-01-01

    Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described. PMID:11238984

  4. Identification and functional analysis of delta-9 desaturase, a key enzyme in PUFA Synthesis, isolated from the oleaginous diatom Fistulifera.

    PubMed

    Muto, Masaki; Kubota, Chihiro; Tanaka, Masayoshi; Satoh, Akira; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

    2013-01-01

    Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ(9) desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ(9) acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ(9) desaturase. Furthermore, the function of these Δ(9) desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ(9) acyl-CoA desaturases showed Δ(9) desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ(9) desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.

  5. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    PubMed

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-asparaginase and L-glutaminase

    PubMed Central

    Shirazian, Pejman; Asad, Sedigheh; Amoozegar, Mohammad Ali

    2016-01-01

    L-asparaginase and L-glutaminase can be effectively used for the treatment of patients who suffer from accute lymphoblastic leukemia and tumor cells. Microbial sources are the best source for the bulk production of these enzymes. However, their long-term administration may cause immunological responses, so screening for new enzymes with novel properties is required. Halophilic and halotolerant bacteria with novel enzymatic characteristics can be considered as a potential source for production of enzymes with different immunological properties. In this study, L-asparaginase and L-glutaminase production by halophilic bacteria isolated from Urmia salt lake was studied. Out of the 85 isolated halophilic and halotolerant bacterial strains, 16 (19 %) showed L-asparaginase activity and 3 strains (3.5 %) showed L-glutaminase activity. Strains with the highest activities were selected for further studies. Based on 16S rDNA sequence analysis, it was shown that the selected isolates for L-asparaginase and L-glutaminase production belong to the genus Bacillus and Salicola, respectively. Both enzymes were produced extracellularly. The strain with the most L-asparaginase production did not show L-glutaminase production which is medically important. The effects of key parameters including temperature, initial pH of the solution, and concentrations of glucose, asparagine or glutamine, and sodium chloride were evaluated by means of response surface methodology (RSM) to optimize enzymes production. Under the obtained optimal conditions, L-asparaginase and L-glutaminase production was increased up to 1.5 (61.7 unit/mL) and 2.6 fold (46.4 unit/mL), respectively. PMID:27330530

  7. The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-asparaginase and L-glutaminase.

    PubMed

    Shirazian, Pejman; Asad, Sedigheh; Amoozegar, Mohammad Ali

    2016-01-01

    L-asparaginase and L-glutaminase can be effectively used for the treatment of patients who suffer from accute lymphoblastic leukemia and tumor cells. Microbial sources are the best source for the bulk production of these enzymes. However, their long-term administration may cause immunological responses, so screening for new enzymes with novel properties is required. Halophilic and halotolerant bacteria with novel enzymatic characteristics can be considered as a potential source for production of enzymes with different immunological properties. In this study, L-asparaginase and L-glutaminase production by halophilic bacteria isolated from Urmia salt lake was studied. Out of the 85 isolated halophilic and halotolerant bacterial strains, 16 (19 %) showed L-asparaginase activity and 3 strains (3.5 %) showed L-glutaminase activity. Strains with the highest activities were selected for further studies. Based on 16S rDNA sequence analysis, it was shown that the selected isolates for L-asparaginase and L-glutaminase production belong to the genus Bacillus and Salicola, respectively. Both enzymes were produced extracellularly. The strain with the most L-asparaginase production did not show L-glutaminase production which is medically important. The effects of key parameters including temperature, initial pH of the solution, and concentrations of glucose, asparagine or glutamine, and sodium chloride were evaluated by means of response surface methodology (RSM) to optimize enzymes production. Under the obtained optimal conditions, L-asparaginase and L-glutaminase production was increased up to 1.5 (61.7 unit/mL) and 2.6 fold (46.4 unit/mL), respectively.

  8. Optimization of Bromelain-Aided Production of Angiotensin I-Converting Enzyme Inhibitory Hydrolysates from Stone Fish Using Response Surface Methodology.

    PubMed

    Muhammad Auwal, Shehu; Zarei, Mohammad; Abdul-Hamid, Azizah; Saari, Nazamid

    2017-03-31

    The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4-7), temperature (40-70 °C), enzyme/substrate (E/S) ratio (0.5%-2%) and time (30-360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries.

  9. Substrate-driven chemotactic assembly in an enzyme cascade.

    PubMed

    Zhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M; Gilson, Michael K; Butler, Peter J; Hess, Henry; Benkovic, Stephen J; Sen, Ayusman

    2018-03-01

    Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

  10. Substrate-driven chemotactic assembly in an enzyme cascade

    NASA Astrophysics Data System (ADS)

    Zhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M.; Gilson, Michael K.; Butler, Peter J.; Hess, Henry; Benkovic, Stephen J.; Sen, Ayusman

    2018-03-01

    Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

  11. Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway.

    PubMed

    Geisler, Christoph; Jarvis, Donald L

    2012-03-02

    Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the

  12. Two key polymorphisms in a newly discovered allele of the Vitis vinifera TPS24 gene are responsible for the production of the rotundone precursor α-guaiene.

    PubMed

    Drew, Damian Paul; Andersen, Trine Bundgaard; Sweetman, Crystal; Møller, Birger Lindberg; Ford, Christopher; Simonsen, Henrik Toft

    2016-02-01

    Rotundone was initially identified as a grape-derived compound responsible for the peppery aroma of Shiraz wine varieties. It has subsequently been found in black and white pepper and several other spices. Because of its potent aroma, the molecular basis for rotundone formation is of particular relevance to grape and wine scientists and industry. We have identified and functionally characterized in planta a sesquiterpene synthase, VvGuaS, from developing grape berries, and have demonstrated that it produces the precursor of rotundone, α-guaiene, as its main product. The VvGuaS enzyme is a novel allele of the sesquiterpene synthase gene, VvTPS24, which has previously been reported to encode VvPNSeInt, an enzyme that produces a variety of selinene-type sesquiterpenes. This newly discovered VvTPS24 allele encodes an enzyme 99.5% identical to VvPNSeInt, with the differences comprising just 6 out of the 561 amino acid residues. Molecular modelling of the enzymes revealed that two of these residues, T414 and V530, are located in the active site of VvGuaS within 4 Å of the binding-site of the substrate, farnesyl pyrophosphate. Mutation of these two residues of VvGuaS into the corresponding polymorphisms in VvPNSeInt results in a complete functional conversion of one enzyme into the other, while mutation of each residue individually produces an intermediate change in the product profile. We have therefore demonstrated that VvGuaS, an enzyme responsible for production of the rotundone precursor, α-guaiene, is encoded by a novel allele of the previously characterized grapevine gene VvTPS24 and that two specific polymorphisms are responsible for functional differences between VvTPS24 alleles. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. Oral enzyme therapy for celiac sprue

    PubMed Central

    Bethune, Michael T; Khosla, Chaitan

    2012-01-01

    Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a lifelong gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically-susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e. glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed. PMID:22208988

  14. Use of a plant-derived enzyme template for the production of the green-note volatile hexanal.

    PubMed

    Schade, Frank; Thompson, John E; Legge, Raymond L

    2003-11-05

    Hexanal is a key organoleptic element of green-note that is found in both fragrances and flavors. We report a novel process for the production of hexanal using immobilized enzyme templates extracted from different plant sources in combination with hollow-fiber ultrafiltration for in situ separation. Enzyme templates, known to be responsible for the synthesis of hexanal from linoleic acid (18:2), were isolated from naturally enriched tissues including carnation petals, strawberry and tomato leaves. These templates were immobilized in an alginate matrix and used as a biocatalyst in a packed-bed bioreactor. Continuous product recovery was achieved using a hollow-fiber ultrafiltration unit. The effects of pH, reaction temperature, and substrate and enzyme concentrations were studied and their effects on hexanal generation identified and optimized. Utilizing optimized conditions, hexanal production 112-fold higher than endogenous steady-state levels in a corresponding amount of plant tissue could be achieved over a 30-minute period. Based on the reactor studies, product inhibition also appears to be an important factor for bioreactor-based hexanal production. Copyright 2003 Wiley Periodicals, Inc.

  15. Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America.

    PubMed

    Ranilla, Lena Galvez; Kwon, Young-In; Apostolidis, Emmanouil; Shetty, Kalidas

    2010-06-01

    Traditionally used medicinal plants, herbs and spices in Latin America were investigated to determine their phenolic profiles, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension. High phenolic and antioxidant activity-containing medicinal plants and spices such as Chancapiedra (Phyllantus niruri L.), Zarzaparrilla (Smilax officinalis), Yerba Mate (Ilex paraguayensis St-Hil), and Huacatay (Tagetes minuta) had the highest anti-hyperglycemia relevant in vitro alpha-glucosidase inhibitory activities with no effect on alpha-amylase. Molle (Schinus molle), Maca (Lepidium meyenii Walp), Caigua (Cyclanthera pedata) and ginger (Zingiber officinale) inhibited significantly the hypertension relevant angiotensin I-converting enzyme (ACE). All evaluated pepper (Capsicum) genus exhibited both anti-hyperglycemia and anti-hypertension potential. Major phenolic compounds in Matico (Piper angustifolium R.), Guascas (Galinsoga parviflora) and Huacatay were chlorogenic acid and hydroxycinnamic acid derivatives. Therefore, specific medicinal plants, herbs and spices from Latin America have potential for hyperglycemia and hypertension prevention associated with Type 2 diabetes. (c) 2010 Elsevier Ltd. All rights reserved.

  16. Industrial Fungal Enzymes: An Occupational Allergen Perspective

    PubMed Central

    Green, Brett J.; Beezhold, Donald H.

    2011-01-01

    Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

  17. A thermal biosensor based on enzyme reaction.

    PubMed

    Zheng, Yi-Hua; Hua, Tse-Chao; Xu, Fei

    2005-01-01

    Application of the thermal biosensor as analytical tool is promising due to advantages as universal, simplicity and quick response. A novel thermal biosensor based on enzyme reaction has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and reference column. The reference column, which is set for eliminating the unspecific heat, is inactived on special enzyme reaction of the ingredient to be detected. The special enzyme reaction takes places in the enzyme reaction column at a constant temperature realizing by a thermoelectric thermostat. Thermal sensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two streams from the enzyme reaction column and the reference column. The analytical example for dichlorvos shows that this biosensor can be used as analytical tool in medicine and biology.

  18. Safety assessment of enzyme-containing personal cleansing products: exposure characterization and development of IgE antibody to enzymes after a 6-month use test.

    PubMed

    Kelling, C K; Bartolo, R G; Ertel, K D; Smith, L A; Watson, D D; Sarlo, K

    1998-02-01

    Enzyme-containing personal cleansing products were being considered for the consumer market. Although enzymes have been marketed safely for many years as ingredients in laundry products, their use in a personal cleansing application represented a new type of exposure for consumers that was not supported by the historical safety data. An exposure assessment and additional safety data would be needed before marketing to ensure consumer safety. The work in this paper was designed to evaluate the potential for inhalation exposure to the enzyme during use of this new product while showering. Then a clinical trial was conducted to determine whether or not the level, duration, and routes of exposure encountered during use of this product would induce a Type I sensitization response to the enzyme. Exposure was assessed during normal showering activities by collecting air samples with both high volume and personal samplers and quantitating enzyme levels with an ELISA. To assess the potential for sensitization, panelists were asked to use a prototype protease-containing bar product for all personal cleansing tasks and to keep a use diary reporting any associated symptoms. Physical and dermatologic examinations and skin prick tests with enzyme were conducted before the test commenced and at 2-month intervals. Exposure assessment results showed that airborne enzyme levels were primarily dependent on the concentration of the enzyme in the personal cleansing product. Mean values for total airborne enzyme protein ranged from 5.7 to 11.8 ng/m3 when enzyme concentration, time of use, and measurement technique remained constant. After 6 months of at-home product use, four of 61 test subjects using the enzyme-containing bar had positive skin prick test responses when tested with the enzyme. The skin prick test data were supplemented with serologic analyses, which detected IgE specific for the protease enzyme. None of these subjects showed any clinical symptoms indicative of allergic

  19. ­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

    PubMed Central

    2018-01-01

    Background Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. Methods To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Results Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Discussion Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia. PMID:29900073

  20. Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

    PubMed

    Marden, James H

    2013-12-01

    Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. © 2013 John Wiley & Sons Ltd.

  1. Consequences of lower food intake on the digestive enzymes activities, the energy reserves and the reproductive outcome in Gammarus fossarum.

    PubMed

    Charron, Laetitia; Geffard, Olivier; Chaumot, Arnaud; Coulaud, Romain; Jaffal, Ali; Gaillet, Véronique; Dedourge-Geffard, Odile; Geffard, Alain

    2015-01-01

    Digestive enzyme activity is often used as a sensitive response to environmental pollution. However, only little is known about the negative effects of stress on digestive capacities and their consequences on energy reserves and reproduction, although these parameters are important for the maintenance of populations. To highlight if changes in biochemical responses (digestive enzymes and reserves) led to impairments at an individual level (fertility), Gammarus fossarum were submitted to a lower food intake throughout a complete female reproductive cycle (i.e. from ovogenesis to offspring production). For both males and females, amylase activity was inhibited by the diet stress, whereas trypsin activity was not influenced. These results underline similar sensitivity of males and females concerning their digestive capacity. Energy reserves decreased with food starvation in females, and remained stable in males. The number of embryos per female decreased with food starvation. Lower digestive activity in males and females therefore appears as an early response. These results underline the ecological relevance of digestive markers, as they make it possible to anticipate upcoming consequences on reproduction in females, a key biological variable for population dynamics.

  2. Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

    NASA Astrophysics Data System (ADS)

    Steen, A. D.

    2015-12-01

    Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

  3. Optimization of Processing Parameters for Extraction of Amylase Enzyme from Dragon (Hylocereus polyrhizus) Peel Using Response Surface Methodology

    PubMed Central

    Abdul Manap, Mohd Yazid; Zohdi, Norkhanani

    2014-01-01

    The main goal of this study was to investigate the effect of extraction conditions on the enzymatic properties of thermoacidic amylase enzyme derived from dragon peel. The studied extraction variables were the buffer-to-sample (B/S) ratio (1 : 2 to 1 : 6, w/w), temperature (−18°C to 25°), mixing time (60 to 180 seconds), and the pH of the buffer (2.0 to 8.0). The results indicate that the enzyme extraction conditions exhibited the least significant (P < 0.05) effect on temperature stability. Conversely, the extraction conditions had the most significant (P < 0.05) effect on the specific activity and pH stability. The results also reveal that the main effect of the B/S ratio, followed by its interaction with the pH of the buffer, was significant (P < 0.05) among most of the response variables studied. The optimum extraction condition caused the amylase to achieve high enzyme activity (648.4 U), specific activity (14.2 U/mg), temperature stability (88.4%), pH stability (85.2%), surfactant agent stability (87.2%), and storage stability (90.3%). PMID:25050403

  4. Homocysteine and the C677T Gene Polymorphism of Its Key Metabolic Enzyme MTHFR Are Risk Factors of Early Renal Damage in Hypertension in a Chinese Han Population

    PubMed Central

    Yun, Lin; Xu, Rui; Li, Guohua; Yao, Yucai; Li, Jiamin; Cong, Dehong; Xu, Xingshun; Zhang, Lihua

    2015-01-01

    Abstract The combined hyperhomocysteinemia condition is a feature of the Chinese hypertensive population. This study used the case-control method to investigate the association between plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme, 5, 10-methylenetetrahydrofolate reductase (MTHFR), and early renal damage in a hypertensive Chinese Han population. A total of 379 adult essential hypertensive patients were selected as the study subjects. The personal information, clinical indicators, and the C677T gene polymorphism of MTHFR were texted. This study used the urine microalbumin/urine creatinine ratio (UACR) as a grouping basis: the hypertension without renal damage group (NRD group) and the hypertension combined with early renal damage group (ERD group). Early renal damage in the Chinese hypertensive population was associated with body weight, systolic pressure, diastolic pressure, urea nitrogen, serum creatinine, cystatin C, uric acid, aldosterone, and glomerular filtration rate. The homocysteine level and the UACR in the TT genotype group were higher than those in the CC genotype group. The binary logistic regression analysis results showed that after sex and age were adjusted, the MTHFR C677T gene polymorphism was correlated with early renal damage in hypertension in both the recessive model and in the additive model. Plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme MTHFR might be independent risk factors of early renal damage in the hypertensive Chinese Han population. PMID:26717388

  5. Homocysteine and the C677T Gene Polymorphism of Its Key Metabolic Enzyme MTHFR Are Risk Factors of Early Renal Damage in Hypertension in a Chinese Han Population.

    PubMed

    Yun, Lin; Xu, Rui; Li, Guohua; Yao, Yucai; Li, Jiamin; Cong, Dehong; Xu, Xingshun; Zhang, Lihua

    2015-12-01

    The combined hyperhomocysteinemia condition is a feature of the Chinese hypertensive population. This study used the case-control method to investigate the association between plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme, 5, 10-methylenetetrahydrofolate reductase (MTHFR), and early renal damage in a hypertensive Chinese Han population.A total of 379 adult essential hypertensive patients were selected as the study subjects. The personal information, clinical indicators, and the C677T gene polymorphism of MTHFR were texted. This study used the urine microalbumin/urine creatinine ratio (UACR) as a grouping basis: the hypertension without renal damage group (NRD group) and the hypertension combined with early renal damage group (ERD group).Early renal damage in the Chinese hypertensive population was associated with body weight, systolic pressure, diastolic pressure, urea nitrogen, serum creatinine, cystatin C, uric acid, aldosterone, and glomerular filtration rate. The homocysteine level and the UACR in the TT genotype group were higher than those in the CC genotype group. The binary logistic regression analysis results showed that after sex and age were adjusted, the MTHFR C677T gene polymorphism was correlated with early renal damage in hypertension in both the recessive model and in the additive model.Plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme MTHFR might be independent risk factors of early renal damage in the hypertensive Chinese Han population.

  6. Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Flounders, A.W.; Carichner, S.C.; Singh, A.K.

    1997-01-01

    Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less

  7. Carboxylesterases: General detoxifying enzymes

    PubMed Central

    Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

    2016-01-01

    Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

  8. Investigating Information Dynamics in Living Systems through the Structure and Function of Enzymes.

    PubMed

    Gatenby, Robert; Frieden, B Roy

    2016-01-01

    Enzymes are proteins that accelerate intracellular chemical reactions often by factors of 105-1012s-1. We propose the structure and function of enzymes represent the thermodynamic expression of heritable information encoded in DNA with post-translational modifications that reflect intra- and extra-cellular environmental inputs. The 3 dimensional shape of the protein, determined by the genetically-specified amino acid sequence and post translational modifications, permits geometric interactions with substrate molecules traditionally described by the key-lock best fit model. Here we apply Kullback-Leibler (K-L) divergence as metric of this geometric "fit" and the information content of the interactions. When the K-L 'distance' between interspersed substrate pn and enzyme rn positions is minimized, the information state, reaction probability, and reaction rate are maximized. The latter obeys the Arrhenius equation, which we show can be derived from the geometrical principle of minimum K-L distance. The derivation is first limited to optimum substrate positions for fixed sets of enzyme positions. However, maximally improving the key/lock fit, called 'induced fit,' requires both sets of positions to be varied optimally. We demonstrate this permits and is maximally efficient if the key and lock particles pn, rn are quantum entangled because the level of entanglement obeys the same minimized value of the Kullback-Leibler distance that occurs when all pn ≈ rn. This implies interchanges pn ⇄ brn randomly taking place during a reaction successively improves key/lock fits, reducing the activation energy Ea and increasing the reaction rate k. Our results demonstrate the summation of heritable and environmental information that determines the enzyme spatial configuration, by decreasing the K-L divergence, is converted to thermodynamic work by reducing Ea and increasing k of intracellular reactions. Macroscopically, enzyme information increases the order in living systems

  9. Engineered control of enzyme structural dynamics and function.

    PubMed

    Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

    2018-04-01

    Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

  10. Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

    PubMed Central

    Ferrer, J.-L.; Austin, M.B.; Stewart, C.; Noel, J.P.

    2010-01-01

    As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4′,5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure–function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution

  11. A Simple Endpoint Assay for Starch-Degrading Enzymes.

    ERIC Educational Resources Information Center

    Kroen, William K.

    1998-01-01

    Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure,…

  12. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

    PubMed Central

    Cantó, Carles; Sauve, Anthony A.; Bai, Peter

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

  13. Evaluation of the energy efficiency of enzyme fermentation by mechanistic modeling.

    PubMed

    Albaek, Mads O; Gernaey, Krist V; Hansen, Morten S; Stocks, Stuart M

    2012-04-01

    Modeling biotechnological processes is key to obtaining increased productivity and efficiency. Particularly crucial to successful modeling of such systems is the coupling of the physical transport phenomena and the biological activity in one model. We have applied a model for the expression of cellulosic enzymes by the filamentous fungus Trichoderma reesei and found excellent agreement with experimental data. The most influential factor was demonstrated to be viscosity and its influence on mass transfer. Not surprisingly, the biological model is also shown to have high influence on the model prediction. At different rates of agitation and aeration as well as headspace pressure, we can predict the energy efficiency of oxygen transfer, a key process parameter for economical production of industrial enzymes. An inverse relationship between the productivity and energy efficiency of the process was found. This modeling approach can be used by manufacturers to evaluate the enzyme fermentation process for a range of different process conditions with regard to energy efficiency. Copyright © 2011 Wiley Periodicals, Inc.

  14. Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

    PubMed Central

    Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling

    2013-01-01

    Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter−1 of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism. PMID:23811508

  15. Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

    PubMed

    Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling; Brautaset, Trygve

    2013-09-01

    Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

  16. The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.

    Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles weremore » significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.« less

  17. Pectin-modifying enzymes and pectin-derived materials: applications and impacts.

    PubMed

    Bonnin, Estelle; Garnier, Catherine; Ralet, Marie-Christine

    2014-01-01

    Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

  18. Biochemistry students' ideas about how an enzyme interacts with a substrate.

    PubMed

    Linenberger, Kimberly J; Bretz, Stacey Lowery

    2015-01-01

    Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

  19. Should we test TPMT enzyme levels before starting azathioprine?

    PubMed

    Richard, Vijay Samuel; Al-Ismail, Deana; Salamat, Ahmed

    2007-08-01

    Thiopurine methyltransferase (TPMT) is the main enzyme responsible for inactivating toxic products of azathioprine (AZA) metabolism. Patients with homozygous deficiency of this enzyme have no enzyme activity and ideally should not be given AZA. Patients with heterozygous deficiency have 50% of enzyme activity and have been shown to respond well and tolerate half a standard dose. We describe a patient with homozygous deficiency of TPMT who developed life threatening neutropenic sepsis, and advocate that all patients should be tested for TPMT activity prior to starting AZA therapy.

  20. In vitro screening and in silico validation revealed key microbes for higher production of significant therapeutic enzyme l-asparaginase.

    PubMed

    Vimal, Archana; Kumar, Awanish

    2017-03-01

    l-asparaginase is an enzyme of medical prominence and reputable as a chemotherapeutic agent. It also has immense potential to cure autoimmune and infectious diseases. The vast application of this enzyme in healthcare sector increases its market demand. However, presently the huge market demand is not achieved completely. This serves the basis to explore better producer microbial strains to bridge the gap between huge demand and supply of this therapeutic enzyme. The present study deals with the successful screening of potent microorganisms producing l-asparaginase. 47 microorganisms were screened including bacteria, fungi, and yeasts. Among all, Penicillium lilacinum showed the highest enzyme activity i.e., 39.67 IU/ml. Shigella flexneri has 23.21 IU/ml of enzyme activity (highest among all the bacterial strain tested). Further, the 3-D structure of l-asparaginase from higher producer strains was developed and validated in silico for its activity. l-asparagine (substrate for l-asparaginase) was docked inside the binding pocket of P. lilacinum and S. flexneri. Docking score for the most common substrate l-asparagine is -6.188 (P. lilacinum), -5.576 (S. flexneri) which is quite good. Moreover, the chemical property of the binding pocket revealed that amino acid residues Phe 243, Gln 260, Gly 365, Asp 386 in P. lilacinum and residues Asp 181, Thr 318, Asn 320 in S. flexneri have an important role in H-bonding. The in silico results supports and strengthen the wet lab results. The outcome obtained motivates to take the present study result from lab to industry for the economic/massive production of this enzyme for the diverse therapeutic application. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Polymer immobilized enzyme optrodes for the detection of penicillin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kulp, T.J.; Camins, I.; Angel, S.M.

    The preparation and performance of two enzyme-based fiber-optic sensors (optrodes) capable of detecting penicillin are described. Each sensor consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme penicillinase and a pH-sensitive fluorescent dye. A signal is produced when the enzyme catalyzes the cleavage of the ..beta..-lactam ring of penicillin to produce penicilloic acid and, consequently, a pH change in the microenvironment of the membrane. The sensors differ in the way the polymer membrane is constructed and in the type of pH indicator dye used. Both optrodes exhibitmore » response times (40-60 s) significantly lower than those of the corresponding enzyme electrodes (2 min). Each gives a linear response over the concentration range of 0.00025 to 0.01 M penicillin G, when measured in a 0.005 M phosphate buffer. The data indicate that these immobilization strategies produce similar results and may be considered complementary alternatives in future enzyme optrode applications.« less

  2. Nonmarket economic user values of the Florida Keys/Key West

    Treesearch

    Vernon R. Leeworthy; J. Michael Bowker

    1997-01-01

    This report provides estimates of the nonmarket economic user values for recreating visitors to the Florida Keys/Key West that participated in natural resource-based activities. Results from estimated travel cost models are presented, including visitor’s responses to prices and estimated per person-trip user values. Annual user values are also calculated and presented...

  3. Nicotinamide N-methyltransferase: more than a vitamin B3 clearance enzyme

    PubMed Central

    Pissios, Pavlos

    2017-01-01

    Nicotinamide N-methyltransferase (NNMT) was originally identified as the enzyme responsible for the methylation of nicotinamide (NAM), one of the forms of vitamin B3. Methylated NAM (MNAM) is eventually excreted from the body. Recent evidence has expanded the role of NNMT beyond clearance of excess vitamin B3. NNMT has been implicated in the regulation of multiple metabolic pathways in tissues such as the adipose tissue and liver, as well as cancer cells, through consumption of methyl donors and generation of active metabolites. This review examines recent findings regarding the function of NNMT in physiology and disease and highlights potential new avenues for therapeutic intervention. Finally, key gaps in our knowledge for this enzymatic system and future areas of investigation are discussed. PMID:28291578

  4. Exquisite Enzyme-Fenton Biomimetic Catalysts for Hydroxyl Radical Production by Mimicking an Enzyme Cascade.

    PubMed

    Zhang, Qi; Chen, Shuo; Wang, Hua; Yu, Hongtao

    2018-03-14

    Hydrogen peroxide (H 2 O 2 ) is a key reactant in the Fenton process. As a byproduct of enzymatic reaction, H 2 O 2 can be obtained via catalytical oxidation of glucose using glucose oxidase in the presence of O 2 . Another oxidation product (gluconic acid) can suitably adjust the microenvironmental pH contributing to the Fe 3+ /Fe 2+ cycle in the Fenton reaction. Enzymes are extremely efficient at catalyzing a variety of reactions with high catalytic activity, substrate specificity, and yields in living organisms. Inspired by the multiple functions of natural multienzyme systems, an exquisite nanozyme-modified α-FeOOH/porous carbon (PC) biomimetic catalyst constructed by in situ growth of glucose oxidase-mimicking Au nanoparticles and crystallization of adsorbed ferric ions within carboxyl into hierarchically PC is developed as an efficient enzyme-Fenton catalyst. The products (H 2 O 2 , ∼4.07 mmol·L -1 ) of the first enzymatic reaction are immediately used as substrates for the second Fenton-like reaction to generate the valuable • OH (∼96.84 μmol·L -1 ), thus mimicking an enzyme cascade pathway. α-FeOOH nanocrystals, attached by C-O-Fe bondings, are encapsulated into the mesoporous PC frameworks, facilitating the electron transfer between α-FeOOH and the PC support and greatly suppressing iron leaching. This study paves a new avenue for designing biomimetic enzyme-based Fenton catalysts mimicking a natural system for • OH production.

  5. From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes

    PubMed Central

    Corcoran, Callan C.; Grady, Cameron R.; Pisitkun, Trairak; Parulekar, Jaya

    2017-01-01

    The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. PMID:27974320

  6. Biogenesis of ER subdomains containing DGAT2, an enzyme involved in industrial oil biosynthesis

    USDA-ARS?s Scientific Manuscript database

    Diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the committed step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl group from the acyl-CoA pool to the sn-3 position of diacylglycerol. The substrate specificity and overall activity of these enzymes play a key role...

  7. Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.

    PubMed

    Visser, Franziska; Müller, Boje; Rose, Judith; Prüfer, Dirk; Noll, Gundula A

    2016-08-09

    The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

  8. Optimization of Bromelain-Aided Production of Angiotensin I-Converting Enzyme Inhibitory Hydrolysates from Stone Fish Using Response Surface Methodology

    PubMed Central

    Auwal, Shehu Muhammad; Zarei, Mohammad; Abdul-Hamid, Azizah; Saari, Nazamid

    2017-01-01

    The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4–7), temperature (40–70 °C), enzyme/substrate (E/S) ratio (0.5%–2%) and time (30–360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries. PMID:28362352

  9. Crystallization and X-ray analysis of 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nango, Eriko; Kumasaka, Takashi, E-mail: tkumasak@bio.titech.ac.jp; Sato, Takao

    2005-07-01

    The crystallization of 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics, is reported. A recombinant 2-deoxy-scyllo-inosose synthase from Bacillus circulans has been crystallized at 277 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.30 Å resolution at 100 K using synchrotron radiation at the Photon Factory. The crystals are monoclinic and belong to space group P2{sub 1}, with unit-cell parameters a = 80.5, b = 70.4, c = 83.0 Å, β = 117.8°. The presence of two molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M})more » of 2.89 Å{sup 3} Da{sup −1} and a solvent constant of 57.4% by volume.« less

  10. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  11. Considerations on the Use of Exogenous Fibrolytic Enzymes to Improve Forage Utilization

    PubMed Central

    Mendoza, Germán D.; Plata-Pérez, Fernando X.

    2014-01-01

    Digestion of cell wall fractions of forage in the rumen is incomplete due to the complex links which limit their degradation. It is therefore necessary to find options to optimize the use of forages in ruminant production systems. One alternative is to use exogenous enzymes. Exogenous fibrolytic enzymes are of fungal or bacterial origin and increase nutrient availability from the cell wall, which consists of three fractions in different proportions depending on the species of forage: digestible, potentially digestible, and indigestible. The response to addition of exogenous enzymes varies with the type of forage; many researchers infer that there are enzyme-forage interactions but fail to explain the biological mechanism. We hypothesize that the response is related to the proportion of the potentially digestible fraction. The exogenous enzyme activity depends on several factors but if the general conditions for enzyme action are available, the potentially digestible fraction may determine the magnitude of the response. Results of experiments with exogenous fibrolytic enzymes in domestic ruminants are inconsistent. This, coupled with their high cost, has made their use unattractive to farmers. Development of cheaper products exploring other microorganisms with fibrolytic activity, such as Fomes fomentarius or Cellulomonas flavigena, is required. PMID:25379525

  12. Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes.

    PubMed

    Serron, S C; Dwivedi, N; Backes, W L

    2000-05-01

    Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to

  13. Gastrointestinal manifestations of Fabry disease: clinical response to enzyme replacement therapy.

    PubMed

    Banikazemi, Maryam; Ullman, Thomas; Desnick, Robert J

    2005-08-01

    Gastrointestinal symptoms are often an early and prominent manifestation of Fabry disease, an X-linked inborn error of metabolism caused by the deficient activity of the lysosomal enzyme, alpha-galactosidase A. This enzyme deficiency results in the progressive accumulation of globotriaosylceramide and other glycosphingolipids in tissue lysosomes throughout the body. In classically affected patients, glycosphingolipid accumulation in the vascular endothelium eventually culminates in life-threatening renal, cardiac, and cerebrovascular disease. In addition, over 50% of patients experience post-prandial abdominal pain and diarrhea that interferes with the ability to work and quality of life. Here, we describe four males aged 17-40 years with classic Fabry disease and severe gastrointestinal symptoms who participated in clinical trials of enzyme replacement therapy with agalsidase beta (Fabrazyme, 1 mg/kg every 2 weeks). Before therapy, the three adult patients experienced post-prandial abdominal pain, bloating, and severe diarrhea with 7-10 bowel movements per day every day and the 17-year-old had weekly episodes of diarrhea with six bowel movements per day. Other symptoms included vomiting, food intolerance, and poor weight gain. All patients took medications for these symptoms (diphenoxylate-atropine [Lomotil], ranitidine hydrochloride [Zantac], or sulfasalazine). After 6-7 months of agalsidase beta therapy, all patients reported "no or only occasional" abdominal pain or diarrhea, had discontinued their gastrointestinal medications, and had gained 3-8 kg. These marked improvements in gastrointestinal symptoms have persisted for over 3 years of treatment. In such patients, enzyme replacement at 1 mg/kg effects an early and significant clinical improvement in the gastrointestinal manifestations of Fabry disease.

  14. Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

    PubMed

    Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

    2016-11-01

    The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Response to angiotensin-converting enzyme inhibition is selectively blunted by high sodium in angiotensin-converting enzyme DD genotype: evidence for gene-environment interaction in healthy volunteers.

    PubMed

    Lely, A Titia; Heerspink, Hiddo J Lambers; Zuurman, Mike; Visser, Folkert W; Kocks, Menno J A; Boomsma, Frans; Navis, Gerjan

    2010-12-01

    Renin-angiotensin-aldosterone system blockade is a cornerstone in cardiovascular protection. Angiotensin-converting enzyme (ACE)-DD genotype has been associated with resistance to angiotensin-converting enzyme inhibition (ACEi), but data are conflicting. As sodium intake modifies the effect of ACEi as well as the genotype-phenotype relationship, we hypothesize gene-environment interaction between sodium-status, the response to ACEi, and ACE genotype. Thirty-five male volunteers (26 ± 9 years; II n = 6, ID n = 18, DD n = 11) were studied during placebo and ACEi (double blind, enalapril 20 mg/day) on low [7 days 50 mmol Na/day (low salt)] and high [7 days 200 mmol Na/day (high salt)] sodium, with a washout of 6 weeks in-between. After each period mean arterial pressure (MAP) was measured before and during graded infusion of angiotensin II (Ang II). During high salt, ACEi reduced MAP in II and ID, but not in DD [II: 88 (78-94) versus 76 (72-88); ID: 87 (84-91) versus 83 (79-87); both P < 0.05 and DD: 86 (82-96) versus 88 (80-90); ns, P < 0.05 between genotypes]. However, during low salt, ACEi reduced MAP in all genotype groups [II: 83 (78-89) versus 77 (72-83); ID: 88 (84-91) versus 82 (78-86); DD: 84 (80-91) versus 81 (75-85); all P < 0.05]. During high salt + ACEi, the Ang II response was blunted in DD, with an 18% rise in MAP during the highest dose versus 22 and 31% in ID and II (P < 0.05). Low salt annihilated these differences. In healthy participants, the MAP response to ACEi is selectively blunted in DD genotype during high salt, accompanied by blunted sensitivity to Ang II. Low salt corrects both abnormalities. Further analysis of this gene-environment interaction in patients may contribute to strategies for improvement of individual treatment efficacy.

  16. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

    NASA Astrophysics Data System (ADS)

    Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

    2014-07-01

    Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

  17. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

    PubMed

    Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

    2014-07-01

    Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

  18. Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    1995-01-01

    A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

  19. Effects of soybean meal on digestive enzymes activity, expression of inflammation-related genes, and chromatin modifications in marine fish (Sparus aurata L.) larvae.

    PubMed

    Perera, Erick; Yúfera, Manuel

    2017-04-01

    The effects of soybean meal (SBM) in early diet of Sparus aurata larvae at two developmental windows were assessed. Prolonged (beyond 14 days post-hatch, dph) feeding with SBM decreased the activity of pancreatic enzymes of larvae. In the absence of SBM these larvae later resumed enzyme activities, but exhibited a significant delay in development. Larvae response to SBM involved up-regulation of extracellular matrix remodeling enzymes and pro-inflammatory cytokines, coupled with a drop in putative intestinal enzymes. Larvae receiving SBM at first feeding appear later to have lower expression of inflammation-related genes, especially those fed SBM until 14 dph. Multivariate analysis confirmed that the duration of the SBM early feeding period drives the physiology of larvae in different directions. Feeding larvae with SBM increased global histone H3 acetylation, whereas upon removal of SBM the process was reverted. A more in deep analysis revealed a dynamic interplay among several reversible histone modifications such as H3K14ac and H3K27m3. Finally, we showed that SBM feeding of larvae results in global hypomethylation that persist after SBM removal. This study is the first demonstrating an effect of diet on marine fish epigenetics. It is concluded that there are limitations for extending SBM feeding of S. aurata larvae beyond 14 dph even under co-feeding with live feed, affecting key physiological processes and normal growth. However, up to 14 dph, SBM does not affect normal development, and produces apparently lasting effects on some key enzymes, genes, and chromatin modifications.

  20. Enzyme-Powered Pumps: From Fundamentals to Applications

    NASA Astrophysics Data System (ADS)

    Ortiz-Rivera, Isamar

    , covering also the effect of the thermodynamics of the enzymatic reaction in the pumping behavior, and (3) the applicability of enzyme pumps as fluid flow-based inhibitor assays and as drug delivery devices. Our findings in each of these areas, gets us closer to our ultimate goal, where we aim to identify the optimal conditions needed for enzyme micropump operation, and construct a general model that could accurately predict enzyme micropump behavior for any enzyme-substrate combination. The information aforementioned has been divided in four chapters. Chapter 1 gives a quick glance into the development of enzyme-powered micropumps: from the systems and observed behaviors inspiring this work, to the first systems that were developed. The stability, duration, and extent of fluid pumping of enzyme pumps in general, are also discussed, along with the optimization of the enzyme-pump design. This chapter aims to provide a general idea of the motivation behind the concept of "enzyme-powered pumps", what are "enzyme-powered pumps", and which are the key features that characterize these systems. Chapter 2 is an extensive analysis of the mechanisms of actuation proposed for enzyme-powered micropumps. This chapter not only covers the first attempts to understand how enzyme pumps work, but also explores further the behavior of urease-powered pumps, which fluid flow patterns cannot be completely predicted only by considering thermal or solutal gradients. The findings of these studies could allow us to rationally control fluid flow for the directed delivery of payloads at designated locations. In Chapters 3 and 4, our focus was to highlight the potential application of enzyme-powered pumps for sensing and delivery. Chapter 3 explores the use of enzyme pumps as fluid flow-based inhibitor assays. At fixed concentrations of an enzyme and its substrate, the presence of an inhibitor can be detected by monitoring the decrease in fluid flow speed. Using this principle, sensors for toxic

  1. Carboxypeptidase M-like enzyme modulates the noncholinergic bronchoconstrictor response in guinea pig.

    PubMed

    Desmazes, N; Lockhart, A; Lacroix, H; Dusser, D J

    1992-11-01

    We studied the effects of aerosolized DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MGTA) (10(-4) M, 90 breaths), a specific inhibitor of carboxypeptidase B-type enzymes, on changes in total pulmonary resistance (RL) induced by aerosolized capsaicin (10(-7) to 10(-4) M; 10 breaths at each concentration) and vagus nerve stimulation (5 V, 5 ms, for 20 s at frequencies varying from 2 to 10 Hz) in anesthetized, atropinized, and ventilated guinea pigs. We also studied the effect of aerosolized MGTA on the bronchoconstrictor response to either aerosolized substance P, neurokinin A (10(-7) to 10(-4) M; 10 breaths at each concentration), and carbachol (10(-5) to 2 x 10(-4) M; 10 breaths at each concentration) or to i.v. administration of neurokinin A (10(-11) to 10(-8) mol/kg), bradykinin (10(-10) to 10(-7) mol/kg), and histamine (10(-8) to 10(-6) mol/kg). Although aerosolized MGTA caused no change in basal RL (P > 0.5), it did potentiate the noncholinergic bronchoconstrictor response to capsaicin (n = 5; P < 0.001) as well as to vagus nerve stimulation (n = 5; P = 0.001). In contrast, MGTA did not potentiate the bronchoconstrictor response to either aerosolized substance P, neurokinin A, and carbachol or to i.v. administration of neurokinin A, histamine, and bradykinin. Carboxypeptidase activity cleaving C-terminal arginine or lysine was found in the membrane preparations of trachea and lung from guinea pigs. The membrane-bound carboxypeptidase activity was maximal at pH 7.0 and was enhanced by the presence of CoCl2 (1 mM) in both the tracheal and lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  3. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    PubMed

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

  4. Physical Unclonable Function Hardware Keys Utilizing Kirchhoff-Law Secure Key Exchange and Noise-Based Logic

    NASA Astrophysics Data System (ADS)

    Kish, Laszlo B.; Kwan, Chiman

    Weak unclonable function (PUF) encryption key means that the manufacturer of the hardware can clone the key but not anybody else. Strong unclonable function (PUF) encryption key means that even the manufacturer of the hardware is unable to clone the key. In this paper, first we introduce an "ultra" strong PUF with intrinsic dynamical randomness, which is not only unclonable but also gets renewed to an independent key (with fresh randomness) during each use via the unconditionally secure key exchange. The solution utilizes the Kirchhoff-law-Johnson-noise (KLJN) method for dynamical key renewal and a one-time-pad secure key for the challenge/response process. The secure key is stored in a flash memory on the chip to provide tamper-resistance and nonvolatile storage with zero power requirements in standby mode. Simplified PUF keys are shown: a strong PUF utilizing KLJN protocol during the first run and noise-based logic (NBL) hyperspace vector string verification method for the challenge/response during the rest of its life or until it is re-initialized. Finally, the simplest PUF utilizes NBL without KLJN thus it can be cloned by the manufacturer but not by anybody else.

  5. Equilibrium softening of an enzyme explored with the DNA spring

    NASA Astrophysics Data System (ADS)

    Tseng, Chiao-Yu; Zocchi, Giovanni

    2014-04-01

    We explore enzyme mechanics using a system of two mechanically coupled biomolecules. Measurements of the mechanical modulation of enzymatic activity in a Luciferase—DNA chimera are presented. These are molecules where the enzyme is deformed by the action of a DNA spring. The response of the enzyme for different states of stress is examined. It is found that small changes in the stress cause large changes in activity. This nonlinear behavior is qualitatively interpreted as arising from a soft regime of the enzyme beyond linear elasticity. This soft regime may enable large conformational motion in enzymes.

  6. High muscle lipid content in obesity is not due to enhanced activation of key triglyceride esterification enzymes or the suppression of lipolytic proteins

    PubMed Central

    Li, Minghua; Paran, Christopher; Wolins, Nathan E.

    2011-01-01

    The mechanisms underlying alterations in muscle lipid metabolism in obesity are poorly understood. The primary aim of this study was to compare the abundance and/or activities of key proteins that regulate intramyocellular triglyceride (IMTG) concentration in the skeletal muscle obtained from obese (OB; n = 8, BMI 38 ± 1 kg/m2) and nonobese (NOB; n = 9, BMI 23 ± 1 kg/m2) women. IMTG concentration was nearly twofold greater in OB vs. NOB subjects (75 ± 15 vs. 40 ± 8 μmol/g dry wt, P < 0.05). In contrast, the activity and protein abundance of key enzymes that regulate the esterification of IMTG (i.e., glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase) were not elevated. We also found no differences between groups in muscle adipose triglyceride lipase and hormone-sensitive lipase (HSL) protein abundance and no differences in phosphorylation of specific sites known to affect HSL activity. However, we did find the elevated IMTG in obesity to be accompanied by a greater abundance of the fatty acid transporter FAT/CD36 in the membrane fraction of muscle from OB vs. NOB subjects (P < 0.05), suggestive of an elevated fatty acid transport capacity. Additionally, protein abundance of the lipid-trafficking protein perilipin 3 was lower (P < 0.05) in muscle from OB vs. NOB when expressed relative to IMTG content. Our findings indicate that the elevated IMTG content found in obese women was not due to an upregulation of key lipogenic proteins or to the suppression of lipolytic proteins. The impact of a low perilipin protein abundance relative to the amount of IMTG in obesity remains to be clarified. PMID:21285405

  7. Weathering of pyrogenic organic matter induces fungal oxidative enzyme response in single culture inoculation experiments.

    PubMed

    Gibson, Christy; Berry, Timothy D; Wang, Ruzhen; Spencer, Julie A; Johnston, Cliff T; Jiang, Yong; Bird, Jeffrey A; Filley, Timothy R

    2016-02-01

    The addition of pyrogenic organic matter (PyOM), the aromatic carbon-rich product of the incomplete combustion of plant biomass or fossil fuels, to soil can influence the rate of microbial metabolism of native soil carbon. The interaction of soil heterotrophs with PyOM may be governed by the surficial chemical and physical properties of PyOM that evolve with environmental exposure. We present results of a 36-day laboratory incubation investigating the interaction of a common white-rot fungus, Trametes versicolor, with three forms of 13 C-enriched (2.08 atom% 13 C) PyOM derived from Pinus ponderosa (450 °C): one freshly produced, and two artificially weathered (254 nm, UV light-water treatment and water-leaching alone). Analysis (FTIR, XPS) of the UV-weathered PyOM showed increased aliphatic C-H content and oxidation of aromatic carbon relative to both the original and water-leached PyOM. The addition of both weathered forms of PyOM stimulated (positively primed) fungal respiration of the growth media, while the unaltered PyOM mildly inhibited (negatively primed) respiration. Artificial weathering resulted in higher oxidative (laccase and peroxidase) enzyme activity than unaltered PyOM, possibly the result of a diminished capacity to bind reactive substrates and extracellular enzymes after weathering. However, and contrary to expectations, simple water-leached weathering resulted in a relatively higher enzyme activity and respiration than that of UV-weathering. The 13 C content of respired CO 2 indicated negligible fungal oxidation of PyOM for all treatments, demonstrating the overall low microbial reactivity of this high temperature PyOM. The increased enzymatic and positive priming response of T. versicolor to weathered PyOM highlights the importance of weathering-induced chemistry in controlling PyOM-microbe-soil carbon interactions.

  8. River Sensitivity and Catchment Connectivity: Key Controls on Geomorphic Response and Effectiveness

    NASA Astrophysics Data System (ADS)

    Lisenby, P.; Fryirs, K.; Croke, J.

    2016-12-01

    The sensitivity of river channels to adjustment and the dynamics of sediment connectivity along a channel network are key controls on the capacity (ability) for a river system to adjust, i.e. the severity, distribution, and type of geomorphic response to disturbance events. In turn, the cumulative impact of geomorphic responses compared with event magnitude will determine the geomorphic effectiveness of a single disturbance event. River sensitivity and sediment connectivity can change significantly over space and time, and vary with changes in internal factors such as channel type and geomorphic landform and external factors such as event sequencing and lithological controls. Correspondingly, the capacity for a geomorphic system to respond to disturbance events will also vary, so that geomorphic effectiveness is not definitively characterized by a static relationship between event magnitude and geomorphic response, but rather is a dynamic comparison between geomorphic response and an actively changing capacity for geomorphic adjustment. Herein, we use the Lockyer Valley, Queensland as a case study to illustrate the variability of river sensitivity and sediment connectivity. We relate this variability to the potential and capacity for geomorphic channel response. We find that the sensitivity to and capacity for geomorphic adjustment varies significantly with channel morphometry and valley position. Additionally, the nature of bedload sediment connectivity changes with the distribution of geomorphic landforms and channel weirs that can impede sediment transference through the system. This variability of river sensitivity and sediment connectivity will control the nature of geomorphic response to disturbance events within the Lockyer Valley. Ultimately, determinations of geomorphic effectiveness for disturbance events will depend on comparisons of their geomorphic impacts with the capacity of the Lockyer geomorphic system to respond.

  9. Enzyme

    MedlinePlus

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

  10. From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

    PubMed

    Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

    2017-03-01

    The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database ( Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. Copyright © 2017 the American Physiological Society.

  11. RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants

    PubMed Central

    Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum; Hong, Jeong Soo; Oh, Tae Rin; Kim, Woo Taek; Yang, Seong Wook

    2017-01-01

    Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. PMID:28712388

  12. C(3)-C(4) Intermediate Species in Alternanthera (Amaranthaceae) : Leaf Anatomy, CO(2) Compensation Point, Net CO(2) Exchange and Activities of Photosynthetic Enzymes.

    PubMed

    Rajendrudu, G; Prasad, J S; Das, V S

    1986-02-01

    Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C(3)-C(4) intermediates based on leaf anatomy, photosynthetic CO(2) compensation point (Gamma), O(2) response of small ghe, Cyrillic, light intensity response of small ghe, Cyrillic, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO(2) compensation points of these two intermediate species at 29 degrees C were much lower than in C(3) plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in small ghe, Cyrillic, the apparent photorespiratory component of O(2) inhibition in A. ficoides is lower than in A. tenella. The small ghe, Cyrillic progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O(2) and 29 degrees C by A. ficoides and A. tenella were 25 to 28 milligrams CO(2) per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C(3) and C(4) species, respectively. The activities of key enzymes of C(4) photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C(3)-C(4) intermediate photosynthesis.

  13. Impact of anti-acidification microbial consortium on carbohydrate metabolism of key microbes during food waste composting.

    PubMed

    Song, Caihong; Li, Mingxiao; Qi, Hui; Zhang, Yali; Liu, Dongming; Xia, Xunfeng; Pan, Hongwei; Xi, Beidou

    2018-07-01

    This study investigated the effect of anti-acidification microbial consortium (AAMC), which act synergistically for rapid bioconversion of organic acids on carbohydrate metabolism of key microbes in the course of food waste (FW) composting by metaproteomics. AAMC was inoculated to the composting mass and compared with treatment with alkaline compounds and the control without any amendment. Inoculating AAMC could effectively accelerate carbohydrate degradation process and improve composting efficiency. Carbohydrate metabolic network profiles showed the inoculation with AAMC could increase significantly the types of enzymes catalysing the degradation of lignin, cellulose and hemicellulose. Furthermore, AAMC inoculum could increase not only diversities of microbes producing key enzymes in metabolism pathways of acetic and propionic acids, but also the amounts of these key enzymes. The increase of diversities of microbes could disperse the pressure from acidic adversity on microorganisms which were capable to degrade acetic and propionic acids. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Effect of posttranslational modifications on enzyme function and assembly.

    PubMed

    Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej

    2013-10-30

    The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Structure, function, and engineering of enzymes in isoflavonoid biosynthesis.

    PubMed

    Wang, Xiaoqiang

    2011-03-01

    Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.

  16. Carbon nanotube-enzyme conjugates for the fabrication of diagnostic biosensors

    NASA Astrophysics Data System (ADS)

    Karunwi, Olukayode Adedamola

    by phage display share some homology with key enzymes (GOx, lactate oxidase and laccase) used in biosensors and enzyme-based biofuel cells. Furthermore, the role of pyrrole electropolymerization as an additive technique for the biofabrication of side-by-side biotransducers for glucose and lactate with minimum cross-talk was investigated along with an electrodeposited layer of Fe/Ni hexacyanoferrate to serve as peroxide mediator, decorated with the electropolymerized PPy-Enzyme biorecognition layer, characterized in vitro, and implanted into the trapezius muscle of a piglet ( Sus scrofa) hemorrhage model. Internal calibration, response under controlled hemorrhage conditions, and post-resection re-characterization were used to evaluate biotransducer performance.

  17. Enzymic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria.

    PubMed

    Roberts, M F

    1981-08-01

    Further studies of the transaminase responsible for the first committed step in alkaloid formation in Conium maculatum have shown the L-alanine: 5-ketooctanal transaminase to occur in both the mitochondria and chloroplast. Experiments suggest that these enzymes are the isoenzymes Transaminase A and B respectively previously isolated by the author. It is suggested that the chloroplast enzyme is normally responsible for alkaloid production.

  18. Response of soil physicochemical properties and enzyme activities to long-term reclamation of coastal saline soil, Eastern China.

    PubMed

    Xie, Xuefeng; Pu, Lijie; Wang, Qiqi; Zhu, Ming; Xu, Yan; Zhang, Meng

    2017-12-31

    Soil enzyme activity during different years of reclamation and land use patterns could indicate changes in soil quality. The objective of this research is to explore the dynamics of 5 soil enzyme activities (dehydrogenase, amylase, urease, acid phosphatase and alkaline phosphatase) involved in C, N, and P cycling and their responses to changes in soil physicochemical properties resulting from long-term reclamation of coastal saline soil. Soil samples from a total of 55 sites were collected from a coastal reclamation area with different years of reclamation (0, 7, 32, 40, 63a) in this study. The results showed that both long-term reclamation and land use patterns have significant effects on soil physicochemical properties and enzyme activities. Compared with the bare flat, soil water content, soil bulk density, pH and electrical conductivity showed a decreasing trend after reclamation, whereas soil organic carbon, total nitrogen and total phosphorus tended to increase. Dehydrogenase, amylase and acid phosphatase activities initially increased and then decreased with increasing years of reclamation, whereas urease and alkaline phosphatase activities were characterized by an increase-decrease-increase trend. Moreover, urease, acid phosphatase and alkaline phosphatase activities exhibited significant differences between coastal saline soil with 63years of reclamation and bare flat, whereas dehydrogenase and amylase activities remained unchanged. Aquaculture ponds showed higher soil water content, pH and EC but lower soil organic carbon, total nitrogen and total phosphorus than rapeseed, broad bean and wheat fields. Rapeseed, broad bean and wheat fields displayed higher urease and alkaline phosphatase activities and lower dehydrogenase, amylase and acid phosphatase activities compared with aquaculture ponds. Redundancy analysis revealed that the soil physicochemical properties explained 74.5% of the variation in soil enzyme activities and that an obvious relationship

  19. Proteomic Profiling of Mitochondrial Enzymes during Skeletal Muscle Aging.

    PubMed

    Staunton, Lisa; O'Connell, Kathleen; Ohlendieck, Kay

    2011-03-07

    Mitochondria are of central importance for energy generation in skeletal muscles. Expression changes or functional alterations in mitochondrial enzymes play a key role during myogenesis, fibre maturation, and various neuromuscular pathologies, as well as natural fibre aging. Mass spectrometry-based proteomics suggests itself as a convenient large-scale and high-throughput approach to catalogue the mitochondrial protein complement and determine global changes during health and disease. This paper gives a brief overview of the relatively new field of mitochondrial proteomics and discusses the findings from recent proteomic surveys of mitochondrial elements in aged skeletal muscles. Changes in the abundance, biochemical activity, subcellular localization, and/or posttranslational modifications in key mitochondrial enzymes might be useful as novel biomarkers of aging. In the long term, this may advance diagnostic procedures, improve the monitoring of disease progression, help in the testing of side effects due to new drug regimes, and enhance our molecular understanding of age-related muscle degeneration.

  20. Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

    2017-01-29

    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Microscale immobilized enzyme reactors in proteomics: latest developments.

    PubMed

    Safdar, Muhammad; Spross, Jens; Jänis, Janne

    2014-01-10

    Enzymatic digestion of proteins is one of the key steps in proteomic analyses. There has been a steady progress in the applied digestion protocols in the past, starting from conventional time-consuming in-solution or in-gel digestion protocols to rapid and efficient methods utilizing different types of microscale enzyme reactors. Application of such microreactors has been proven beneficial due to lower sample consumption, higher sensitivity and straightforward coupling with LC-MS set-ups. Novel stationary phases, immobilization techniques and device formats are being constantly developed and tested to optimize digestion efficiency of proteolytic enzymes. This review focuses on the latest developments associated with the preparation and application of microscale enzyme reactors for proteomics applications since 2008 onwards. A special attention has been paid to the discussion of different stationary phases applied for immobilization purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. New tricks for the glycyl radical enzyme family

    PubMed Central

    Backman, Lindsey R.F.; Funk, Michael A.; Dawson, Christopher D.; Drennan, Catherine. L.

    2018-01-01

    Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction, and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O, and C-N bond breaking and formation steps that are otherwise challenging for non-radical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs. PMID:28901199

  3. Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter

    PubMed Central

    Huang, Yuhong; Yi, Zhuolin; Jin, Yanling; Huang, Mengjun; He, Kaize; Liu, Dayu; Luo, Huibo; Zhao, Dong; He, Hui; Fang, Yang; Zhao, Hai

    2017-01-01

    Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products. PMID:28955318

  4. Enzymes, detergents and skin: facts and fantasies.

    PubMed

    Basketter, D A; English, J S C; Wakelin, S H; White, I R

    2008-06-01

    In their raw state, enzymes of bacterial/fungal origin cause allergic reactions in the lung. Proteolytic enzymes also cause irritation to skin, eyes and the respiratory tract. For 40 years, encapsulated enzymes have been used worldwide in detergent products, especially laundry formulations, and have increasing importance due to biodegradability and functionality at low temperatures, offering environmental benefits. Uniquely to the U.K., for years it has been suggested that the inclusion of enzymes in such products leads to adverse skin reactions, including erythema, pruritus and exacerbation of eczema. In this review, we look at the facts, asking whether there is evidence that the hazards identified for enzymes translate into any risk for consumer health. By considering the actual exposures in consumer use and exaggerated product usage, it is concluded that the irritating and allergenic hazards of enzyme raw materials do not translate into a risk of skin reactions, either irritant or allergic. Investigations of numerous individuals with skin complaints attributed to laundry products demonstrate convincingly that enzymes were not responsible. Indeed, enzyme-containing laundry products have an extensive history of safe use. Thus, the supposed adverse effects of enzymes on skin seem to be a consequence of a mythology. The important practical lesson is that when primary or secondary care practitioners are presented with a skin complaint, it should not be dismissed as a result of using an enzyme-containing laundry product as the diagnosis will certainly lie elsewhere. Education for healthcare professionals could usefully be enhanced to take this on board.

  5. Redox regulation of carbon storage and partitioning in response to light and sugars.

    PubMed

    Geigenberger, Peter; Kolbe, Anna; Tiessen, Axel

    2005-06-01

    Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid

  6. Spatial distribution of enzyme driven reactions at micro-scales

    NASA Astrophysics Data System (ADS)

    Kandeler, Ellen; Boeddinghaus, Runa; Nassal, Dinah; Preusser, Sebastian; Marhan, Sven; Poll, Christian

    2017-04-01

    Studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of soil microorganisms that dominate in natural environments. In comparison with aquatic systems, soils are particularly heterogeneous. Soil heterogeneity results from the interaction of a hierarchical series of interrelated variables that fluctuate at many different spatial and temporal scales. Whereas spatial dependence of chemical and physical soil properties is well known at scales ranging from decimetres to several hundred metres, the spatial structure of soil enzymes is less clear. Previous work has primarily focused on spatial heterogeneity at a single analytical scale using the distribution of individual cells, specific types of organisms or collective parameters such as bacterial abundance or total microbial biomass. There are fewer studies that have considered variations in community function and soil enzyme activities. This presentation will give an overview about recent studies focusing on spatial pattern of different soil enzymes in the terrestrial environment. Whereas zymography allows the visualization of enzyme pattern in the close vicinity of roots, micro-sampling strategies followed by MUF analyses clarify micro-scale pattern of enzymes associated to specific microhabitats (micro-aggregates, organo-mineral complexes, subsoil compartments).

  7. Rational assignment of key motifs for function guides in silico enzyme identification.

    PubMed

    Höhne, Matthias; Schätzle, Sebastian; Jochens, Helge; Robins, Karen; Bornscheuer, Uwe T

    2010-11-01

    Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

  8. Degradative enzymes modulate airway responses to intravenous neurokinins A and B.

    PubMed

    Shore, S A; Drazen, J M

    1989-12-01

    We studied the effects of the neutral endopeptidase (NEP) inhibitor thiorphan (1.7 mg/kg iv) and the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg iv) on airway responses to rapid intravenous infusions of neurokinin A (NKA) and neurokinin B (NKB) in anesthetized, mechanically ventilated guinea pigs. The dose of NKA required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) was fivefold less (P less than 0.0001) in animals treated with thiorphan compared with controls. NKA1-8, a product resulting from cleavage of NKA by NEP, had no bronchoconstrictor activity. Similar results were obtained by using NKB as the bronchoconstricting agent. Captopril had no significant effect on airway responses to NKA or NKB. In contrast, both thiorphan and captopril decrease the ED50GL for substance P (SP). We also compared the relative bronchoconstrictor potency of NKA, NKB, and SP. In control animals, the rank order of ED50GL values was NKA much less than NKB = SP. NKA also caused a more prolonged bronchoconstriction than SP or NKB. Thiorphan had no effect on the rank order of bronchoconstrictor potency, but in animals treated with captopril, the rank order of ED50GL values was altered to NKA less than SP less than NKB. These results suggest that degradation of NKA and NKB by NEP but not by ACE is an important determinant of the bronchoconstriction induced by these peptides. The degradation by ACE of SP but not NKA or NKB influences the observed relative potency of the three tachykinins as bronchoactive agents.

  9. Sensitivity of bacterioplankton nitrogen metabolism to eutrophication in sub-tropical coastal waters of Key West, Florida.

    PubMed

    Hoch, Matthew P; Dillon, Kevin S; Coffin, Richard B; Cifuentes, Luis A

    2008-05-01

    Expression of intracellular ammonium assimilation enzymes were used to assess the response of nitrogen (N) metabolism in bacterioplankton to N-loading of sub-tropical coastal waters of Key West, Florida. Specific activities of glutamine synthetase (GS) and total glutamate dehydrogenase (GDHT) were measured on the bacterial size fraction (<0.8 microm) to assess N-deplete versus N-replete metabolic states, respectively. Enzyme results were compared to concentrations of dissolved organic matter and nutrients and to the biomass and production of phytoplankton and bacteria. Concentrations of dissolved inorganic N (DIN), dissolved organic N (DON), and dissolved organic carbon (DOC) positively correlated with specific activities of GDHT and negatively correlated with that of GS. Total dissolved N (TDN) concentration explained 81% of variance in bacterioplankton GDHT:GS activity ratio. The GDHT:GS ratio, TDN, DOC, and bacterial parameters decreased in magnitude along a tidally dynamic trophic gradient from north of Key West to south at the reef tract, which is consistent with the combined effects of localized coastal eutrophication and tidal exchange of seawater from the Southwest Florida Shelf and Florida Strait. The N-replete bacterioplankton north of Key West can regenerate ammonium which sustains primary production transported south to the reef. The range in GDHT:GS ratios was 5-30 times greater than that for commonly used indicators of planktonic eutrophication, which emphasizes the sensitivity of bacterioplankton N-metabolism to changes in N-bioavailability caused by nutrient pollution in sub-tropical coastal waters and utility of GDHT:GS ratio as an bioindicator of N-replete conditions.

  10. RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants.

    PubMed

    Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum; Hong, Jeong Soo; Oh, Tae Rin; Kim, Woo Taek; Yang, Seong Wook

    2017-08-01

    Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. [BMB Reports 2017; 50(8): 393-400].

  11. Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

    PubMed

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

    Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

  12. Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad: recent developments in enzymology and modeling studies.

    PubMed

    Bruijnincx, Pieter C A; van Koten, Gerard; Klein Gebbink, Robertus J M

    2008-12-01

    Iron-containing enzymes are one of Nature's main means of effecting key biological transformations. The mononuclear non-heme iron oxygenases and oxidases have received the most attention recently, primarily because of the recent availability of crystal structures of many different enzymes and the stunningly diverse oxidative transformations that these enzymes catalyze. The wealth of available structural data has furthermore established the so-called 2-His-1-carboxylate facial triad as a new common structural motif for the activation of dioxygen. This superfamily of mononuclear iron(ii) enzymes catalyzes a wide range of oxidative transformations, ranging from the cis-dihydroxylation of arenes to the biosynthesis of antibiotics such as isopenicillin and fosfomycin. The remarkable scope of oxidative transformations seems to be even broader than that associated with oxidative heme enzymes. Not only are many of these oxidative transformations of key biological importance, many of these selective oxidations are also unprecedented in synthetic organic chemistry. In this critical review, we wish to provide a concise background on the chemistry of the mononuclear non-heme iron enzymes characterized by the 2-His-1-carboxylate facial triad and to discuss the many recent developments in the field. New examples of enzymes with unique reactivities belonging to the superfamily have been reported. Furthermore, key insights into the intricate mechanistic details and reactive intermediates have been obtained from both enzyme and modeling studies. Sections of this review are devoted to each of these subjects, i.e. the enzymes, biomimetic models, and reactive intermediates (225 references).

  13. Cystathionine metabolic enzymes play a role in the inflammation resolution of human keratinocytes in response to sub-cytotoxic formaldehyde exposure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Eunyoung

    Low-level formaldehyde exposure is inevitable in industrialized countries. Although daily-life formaldehyde exposure level is practically impossible to induce cell death, most of mechanistic studies related to formaldehyde toxicity have been performed in cytotoxic concentrations enough to trigger cell death mechanism. Currently, toxicological mechanisms underlying the sub-cytotoxic exposure to formaldehyde are not clearly elucidated in skin cells. In this study, the genome-scale transcriptional analysis in normal human keratinocytes (NHKs) was performed to investigate cutaneous biological pathways associated with daily life formaldehyde exposure. We selected the 175 upregulated differentially expressed genes (DEGs) and 116 downregulated DEGs in NHKs treated with 200 μMmore » formaldehyde. In the Gene Ontology (GO) enrichment analysis of the 175 upregulated DEGs, the endoplasmic reticulum (ER) unfolded protein response (UPR) was identified as the most significant GO biological process in the formaldeyde-treated NHKs. Interestingly, the sub-cytotoxic formaldehyde affected NHKs to upregulate two enzymes important in the cellular transsulfuration pathway, cystathionine γ-lyase (CTH) and cystathionine-β-synthase (CBS). In the temporal expression analysis, the upregulation of the pro-inflammatory DEGs such as MMP1 and PTGS2 was detected earlier than that of CTH, CBS and other ER UPR genes. The metabolites of CTH and CBS, L-cystathionine and L-cysteine, attenuated the formaldehyde-induced upregulation of pro-inflammatory DEGs, MMP1, PTGS2, and CXCL8, suggesting that CTH and CBS play a role in the negative feedback regulation of formaldehyde-induced pro-inflammatory responses in NHKs. In this regard, the sub-cytotoxic formaldehyde-induced CBS and CTH may regulate inflammation fate decision to resolution by suppressing the early pro-inflammatory response. - Highlights: • Sub-cytotoxic formaldehyde upregulates ER UPR-associated genes in NHKs. • Formaldehyde

  14. Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

    PubMed

    Bate, Paul; Warwicker, Jim

    2004-07-02

    Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

  15. Key experiences of community engagement and social mobilization in the Ebola response.

    PubMed

    Laverack, Glenn; Manoncourt, Erma

    2016-03-01

    The ongoing outbreak of the Ebola virus in West Africa is the largest on record; it has undermined already fragile healthcare systems and presented new challenges to contain the spread of the disease. Based on our observations in the field and insights from referenced sources, we aimed to identify key experiences of community engagement and social mobilization efforts in the current Ebola response. We concluded that there is no excuse not to actively involve local people and that the United Nations (UN) agencies and other partners did learn from their earlier mistakes to make a genuine attempt to better engage with communities. However, bottom-up approaches have not been widely implemented during the response and the reasons for not doing so must be further assessed. Health promotion can make an important contribution, because it shows how to enable people to take more control over their lives and health. This commentary can provide a guide to agencies to understand an appropriate way forward when the next Ebola outbreak inevitably occurs. © The Author(s) 2015.

  16. Green tea polyphenol (−)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: Responses of major antioxidant enzymes and the Nrf2 rescue pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Dongxu; Wang, Yijun; Wan, Xiaochun

    (−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been suggested to have numerous health-promoting effects. On the other hand, high-dose EGCG is able to evoke hepatotoxicity. In the present study, we elucidated the responses of hepatic major antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2) rescue pathway to high-dose levels of EGCG in Kunming mice. At a non-lethal toxic dose (75 mg/kg, i.p.), repeated EGCG treatments markedly decreased the levels of superoxide dismutase, catalase, and glutathione peroxidase. As a rescue response, the nuclear distribution of Nrf2 was significantly increased; a battery of Nrf2-target genes, including heme oxygenase 1more » (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and those involved in glutathione and thioredoxin systems, were all up-regulated. At the maximum tolerated dose (45 mg/kg, i.p.), repeated EGCG treatments did not disturb the major antioxidant defense. Among the above-mentioned genes, only HO1, NQO1, and GST genes were significantly but modestly up-regulated, suggesting a comprehensive and extensive activation of Nrf2-target genes principally occurs at toxic levels of EGCG. At a lethal dose (200 mg/kg, i.p.), a single EGCG treatment dramatically decreased not only the major antioxidant defense but also the Nrf2-target genes, demonstrating that toxic levels of EGCG are able to cause a biphasic response of Nrf2. Overall, the mechanism of EGCG-triggered hepatotoxicity involves suppression of major antioxidant enzymes, and the Nrf2 rescue pathway plays a vital role for counteracting EGCG toxicity. - Highlights: • EGCG at maximum tolerated dose does not disturb hepatic major antioxidant defense. • EGCG at maximum tolerated dose modestly upregulates hepatic Nrf2 target genes. • EGCG at toxic dose suppresses hepatic major antioxidant enzymes. • EGCG at non-lethal toxic dose pronouncedly activates hepatic Nrf2 rescue response. • EGCG

  17. Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability.

    PubMed

    Karmali, A; Pacheco, R; Tata, R; Brown, P

    2001-03-01

    Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

  18. Enzyme cofactors: Double-edged sword for catalysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Ivaylo

    2013-01-01

    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  19. Isoquercetin ameliorates hyperglycemia and regulates key enzymes of glucose metabolism via insulin signaling pathway in streptozotocin-induced diabetic rats.

    PubMed

    Jayachandran, Muthukumaran; Zhang, Tongze; Ganesan, Kumar; Xu, Baojun; Chung, Stephen Sum Man

    2018-06-15

    Among the foremost common flavonoids within the human diet, quercetin glycosides possess neuroprotective, cardioprotective, anti-oxidative, chemopreventive, and anti-allergic properties. Isoquercetin is one such promising candidate with anti-diabetic potential. However, complete studies of its molecular action on insulin signaling pathway and carbohydrate metabolizing enzymes remain unclear. Hence, we have designed this study to accumulate the experimental evidence in support of anti-diabetic effects of isoquercetin. Male albino Wistar rats were divided into seven groups. Rats (Groups 3-7) were administered a single intraperitoneal injection of streptozotocin (STZ; 40 mg/kg b.w) to induce diabetes mellitus. As an extension, STZ rats received isoquercetin at three different doses (20, 40 and 80 mg/kg b.w), and Group 7 rats received glibenclamide (standard drug) (600 μg/kg b.w). The results showed that STZ exaggerated blood sugar, decreased insulin, altered metabolizing enzymes, and impaired the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. Supplementation with isoquercetin significantly normalized blood sugar levels, insulin and regulated the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. The results achieved with isoquercetin are similar to that of standard drug glibenclamide. The findings suggest isoquercetin could be a possible therapeutic agent for treating diabetes mellitus in the near future. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  1. Enzyme exposure in the British baking industry.

    PubMed

    Elms, J; Robinson, E; Mason, H; Iqbal, S; Garrod, A; Evans, G S

    2006-06-01

    Enzymes are commonly used in the baking industry, as they can improve dough quality and texture and lengthen the shelf life of the final product. There is little published information highlighting exposure to enzymes (other than fungal alpha-amylase) in the baking industry, therefore the purpose of this study was to identify antibodies and develop assays for the measurement of a variety of such enzymes in samples of airborne flour dust. Polyclonal antibodies to bacterial amylase, glucose oxidase and amyloglucosidase were identified and developed into ELISA assays. The assays showed limited cross-reactivity with other enzymes commonly used in the baking industry. We measured levels of airborne enzymes in 195 personal air samples taken from a sample of 55 craft baking establishments. We were able to detect amyloglucosidase in 9% (16/184) of the samples, fungal alpha-amylase in 6% (11/171), bacterial alpha-amylase in 7% (13/195). However, we were unable to detect glucose oxidase in any of the samples. Measurements for protease enzymes were not carried out. Median levels in detectable samples of amyloglucosidase, fungal alpha-amylase and bacterial amylase were similar at 10.3, 5.3 and 5.9 ng/m(3), respectively. These figures represent the total enzyme protein (active and inactive) measured. There are few data in the literature regarding sensitization and exposure-response relationships to these enzymes, and indeed there is often a lack of information within the industry as to the precise enzyme content of particular baking ingredients. As a precautionary measure, all enzymes are regarded as having the potential to cause respiratory sensitization. Consequently, exposures need to be controlled to as low a level as reasonably practicable, and future investigation may highlight the importance of measuring a variety of enzyme exposures and standardizing these methodologies to inform approaches to adequate control.

  2. Metaproteomic Analysis of a Chemosynthetic Hydrothermal Vent Community Reveals Insights into Key-Metabolic Processes

    NASA Astrophysics Data System (ADS)

    Steen, I.; Stokke, R.; Lanzen, A.; Pedersen, R.; Øvreås, L.; Urich, T.

    2010-12-01

    internal sulfur cycle within the community. The community contained expressed enzymes of a variety of carbon metabolism pathways. Key enzymes of the reverse TCA cycle for fixation of CO2 and the Wood-Ljungdahl pathway for oxidation of acetyl-CoA and / or the fixation of CO2 were found. Key enzymes of aerobic and anaerobic methane-oxidation pathways were identified as well, namely particulate methane monooxygenase and methyl-Coenzyme M reductase. Various house-keeping gene-products, like cold- and heat shock proteins as well as ribosomal proteins and ATP synthases were identified. This approach has a future potential of broadening our understanding of environmental complexity and regulation in response to geochemical constraints. [1] Dupierris, V., Masselon, C., Court, M., Kieffer-Jaquinod, S., and Bruley, C. (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25, 1980-1981.

  3. Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

    NASA Astrophysics Data System (ADS)

    Subramanian, Periasamy; Bupesh, Giridharan

    2011-06-01

    Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

  4. The linkage disequilibrium pattern of the angiotensin converting enzyme gene in Arabic and Asian population groups.

    PubMed

    Kharrat, Najla; Abdelmouleh, Wafa; Abdelhedi, Rania; Alfadhli, Suad; Rebai, Ahmed

    2012-01-01

    DNA variations within the Angiotensin-Converting Enzyme (ACE) gene have been shown to be involved in the aetiology of several common diseases and the therapeutic response. This study reports a comparison of haplotype analysis of five SNPs in the ACE gene region using a sample of 100 healthy subjects derived from five different populations (Tunisian, Iranian, Kuwaiti, Bahraini and Indian). Strong linkage disequilibrium was found among all SNPs studied for all populations. Two SNPs (rs1800764 and rs4340) were identified as key SNPs for all populations. These SNPs will be valuable for future effective association studies of the ACE gene polymorphisms in Arab and Asian populations.

  5. Inhibitory Activities of Zygophyllum album: A Natural Weight-Lowering Plant on Key Enzymes in High-Fat Diet-Fed Rats

    PubMed Central

    Mnafgui, Kais; Hamden, Khaled; Ben Salah, Hichem; Kchaou, Mouna; Nasri, Mbarek; Slama, Sadok; Derbali, Fatma; Allouche, Noureddine; Elfeki, Abdelfattah

    2012-01-01

    Obesity is a serious health problem that increased risk for many complications, including diabetes and cardiovascular disease. The results showed EZA, which found rich in flavonoids and phenolic compounds, exhibited an inhibitory activity on pancreatic lipase in vitro with IC50 of 91.07 μg/mL. In vivo administration of this extract to HFD-rats lowered body weight and serum leptin level; and inhibited lipase activity of obese rats by 37% leading to notable decrease of T-Ch, TGs and LDL-c levels accompanied with an increase in HDL-c concentration in serum and liver of EZA treated HFD-rats. Moreover, the findings revealed that EZA helped to protect liver tissue from the appearance of fatty cysts. Interestingly, supplementation of EZA modulated key enzyme related to hypertension such as ACE by 36% in serum of HFD animals and improve some of serum electrolytes such as Na+, K+, Cl−, Ca2+ and Mg2+. Moreover, EZA significantly protected the liver-kidney function by reverted back near to normal the values of the liver-kidney dysfunction indices AST&ALT, ALP, CPK and GGT activities, decreased T-Bili, creat, urea and uric acid rates. In conclusion, these results showed a strong antihypelipidemic effect of EZA which can delay the occurrence of dislipidemia and hypertension. PMID:23258993

  6. Differential antioxidative enzyme responses of Jatropha curcas L. to chromium stress.

    PubMed

    Yadav, Santosh Kumar; Dhote, Monika; Kumar, Phani; Sharma, Jitendra; Chakrabarti, Tapan; Juwarkar, Asha A

    2010-08-15

    Chromium (Cr) tolerant and accumulation capability of Jatropha curcas L. was tested in Cr spiked soil amended with biosludge and biofertilizer. Plants were cultivated in soils containing 0, 25, 50, 100 and 250 mg kg(-1) of Cr for one year with and without amendment. Plant tissue analysis showed that combined application of biosludge and biofertilizer could significantly reduce Cr uptake and boost the plant biomass, whereas biofertilizer alone did not affect the uptake and plant growth. Antioxidative responses of catalase (CAT), ascorbate peroxidase (APX) and glutathione S-transferase (GST) were increased with increasing Cr concentration in plant. Hyperactivity of the CAT and GST indicated that antioxidant enzymes played an important role in protecting the plant from Cr toxicity. However, APX took a little part in detoxification of H(2)O(2) due to its sensitivity to Cr. Therefore, reduced APX activity was recorded. Reduced glutathione (GSH) activity was recorded in plant grown on/above 100 mg kg(-1) of Cr in soil. The study concludes that J. curcas could grow under chromium stress. Furthermore, the results encouraged that J. curcas is a suitable candidate for the restoration of Cr contaminated soils with the concomitant application of biosludge and biofertilizer. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

    PubMed

    Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

    2018-04-03

    5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Establishing Chinese medicine characteristic tumor response evaluation system is the key to promote internationalization of Chinese medicine oncology.

    PubMed

    Li, Jie; Li, Lei; Liu, Rui; Lin, Hong-sheng

    2012-10-01

    The features and advantages of Chinese medicine (CM) in cancer comprehensive treatment have been in the spotlight of experts both at home and abroad. However, how to evaluate the effect of CM more objectively, scientifically and systematically is still the key problem of clinical trial, and also a limitation to the development and internationalization of CM oncology. The change of tumor response evaluation system in conventional medicine is gradually consistent with the features of CM clinical effect, such as they both focus on a combination of soft endpoints (i.e. quality of life, clinical benefit, etc.) and hard endpoints (i.e. tumor remission rate, time to progress, etc.). Although experts have proposed protocols of CM tumor response evaluation criteria and come to an agreement in general, divergences still exist in the importance, quantification and CM feature of the potential endpoints. Thus, establishing a CM characteristic and wildly accepted tumor response evaluation system is the key to promote internationalization of CM oncology, and also provides a more convenient and scientific platform for CM international cooperation and communication.

  9. Functional enzyme-based modeling approach for dynamic simulation of denitrification process in hyporheic zone sediments: Genetically structured microbial community model

    NASA Astrophysics Data System (ADS)

    Song, H. S.; Li, M.; Qian, W.; Song, X.; Chen, X.; Scheibe, T. D.; Fredrickson, J.; Zachara, J. M.; Liu, C.

    2016-12-01

    Modeling environmental microbial communities at individual organism level is currently intractable due to overwhelming structural complexity. Functional guild-based approaches alleviate this problem by lumping microorganisms into fewer groups based on their functional similarities. This reduction may become ineffective, however, when individual species perform multiple functions as environmental conditions vary. In contrast, the functional enzyme-based modeling approach we present here describes microbial community dynamics based on identified functional enzymes (rather than individual species or their groups). Previous studies in the literature along this line used biomass or functional genes as surrogate measures of enzymes due to the lack of analytical methods for quantifying enzymes in environmental samples. Leveraging our recent development of a signature peptide-based technique enabling sensitive quantification of functional enzymes in environmental samples, we developed a genetically structured microbial community model (GSMCM) to incorporate enzyme concentrations and various other omics measurements (if available) as key modeling input. We formulated the GSMCM based on the cybernetic metabolic modeling framework to rationally account for cellular regulation without relying on empirical inhibition kinetics. In the case study of modeling denitrification process in Columbia River hyporheic zone sediments collected from the Hanford Reach, our GSMCM provided a quantitative fit to complex experimental data in denitrification, including the delayed response of enzyme activation to the change in substrate concentration. Our future goal is to extend the modeling scope to the prediction of carbon and nitrogen cycles and contaminant fate. Integration of a simpler version of the GSMCM with PFLOTRAN for multi-scale field simulations is in progress.

  10. Roles and responsibilities of pharmacists with respect to natural health products: key informant interviews.

    PubMed

    Olatunde, Shade; Boon, Heather; Hirschkorn, Kristine; Welsh, Sandy; Bajcar, Jana

    2010-03-01

    Although many pharmacies sell natural health products (NHPs), there is no clear definition as to the roles and responsibilities (if any) of pharmacists with respect to these products. The purpose of this study was to explore pharmacy and stakeholder leaders' perceptions of pharmacists' professional NHP roles and responsibilities. Semi-structured key informant interviews were conducted with pharmacy leaders (n=17) and stakeholder (n=18) leaders representing consumers, complementary and alternative medicine practitioners, conventional health care practitioners, and industry across Canada. All participants believed a main NHP responsibility for pharmacists was in safety monitoring, although a one challenge identified in the interviews was pharmacists' general lack of NHP knowledge; however, stakeholder leaders did not expect pharmacists to be experts, but should have a basic level of knowledge about NHPs. Participants described pharmacists' professional roles and responsibilities for NHPs as similar to those for over-the-counter drugs; more awareness of existing NHP-related pharmacy policies is needed, and pharmacy owners/managers should provide additional training to ensure front-line pharmacists have appropriate knowledge of NHPs sold in the pharmacy. Copyright 2010 Elsevier Inc. All rights reserved.

  11. Responses of soil enzyme activity and microbial community compositions to nitrogen addition in bulk and microaggregate soil in the temperate steppe of Inner Mongolia

    NASA Astrophysics Data System (ADS)

    Shi, Yao; Sheng, Lianxi; Wang, Zhongqiang; Zhang, Xinyu; He, Nianpeng; Yu, Qiang

    2016-10-01

    In order to explore the responses of soil enzyme activities and microbial community compositions to long-term nitrogen (N) addition in both bulk soil and microaggregate of chestnut soil, we conducted a 7-year urea addition experiment with N treatments at 6 levels (0, 56, 112, 224, 392 and 560 kg N ha-1 yr-1) in a temperate steppe of Inner Mongolia in China. Soil properties and the activities of four enzymes involved in carbon (C), nitrogen (N) and phosphorus (P) cycling were measured in both bulk soil and microaggregate, and phospholipid fatty acids (PLFAs) were measured in bulk soil. The results indicated that: 1) in bulk soil, N addition significantly decreased β-1,4-glucosidase (BG) and leucine aminopeptidase (LAP) activities at the treatment amounts of 224, 392 and 560 kg N ha-1 yr-1, and obviously suppressed β-1,4-N-acetylglucosaminidase (NAG) activity at the treatment amount of 560 kg N ha-1 yr-1. N addition enhanced total PLFAs (totPLFAs) and bacterial PLFAs (bacPLFAs) at the treatment amounts of 392 and 560 kg N ha-1 yr-1, respectively, but fungal PLFAs showed no response to N addition. The activities of BG, NAG and LAP were positively correlated with soil pH, but negatively correlated with the concentration of NH 4 + -N; 2) in microaggregate (53-250 μm), the activities of BG, NAG and AP showed no response to increased addition of N, but the significantly decreased LAP activity was observed at the treatment amount of 392 kg N ha-1 yr-1. These results suggested that enzyme activities were more sensitive to N addition than PLFA biomarkers in soil, and LAP activity in microaggregate may be a good indicator for evaluating N cycle response to long-term N addition.

  12. The structure and function of Alzheimer's gamma secretase enzyme complex.

    PubMed

    Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

    2009-01-01

    The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.

  13. Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

    PubMed

    Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F

    2018-04-17

    Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

  14. CAH1 and CAH2 as key enzymes required for high bicarbonate tolerance of a novel microalga Dunaliella salina HTBS.

    PubMed

    Hou, Yuyong; Liu, Zhiyong; Zhao, Yue; Chen, Shulin; Zheng, Yubin; Chen, Fangjian

    2016-06-01

    Outdoor microalgal cultivation with high concentration bicarbonate has been considered as a strategy for reducing contamination and improving carbon supply efficiency. The mechanism responsible for algae's strong tolerance to high bicarbonate however, remains not clear. In this study, we isolated and characterized a strain and revealed its high bicarbonate tolerant mechanism by analyzing carbonic anhydrase (CA). The strain was identified as Dunaliella salina HTBS with broad temperature adaptability (7-30°C). The strain grew well under 30% CO2 or 70gL(-1) NaHCO3. In comparison, two periplasm CAs (CAH1 and CAH2) were detected with immunoblotting analysis in HTBS but not in a non-HCO3(-)-tolerant strain. The finding was also verified by an enzyme inhibition assay in which only HTBS showed significant inhibition by extracellular CA inhibitor. Thus, we inferred that the extracellular CAH1 and CAH2 played a multifunctional role in the toleration of high bicarbonate by HTBS. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Responses of non-starch polysaccharide-degrading enzymes on digestibility and performance of growing pigs fed a diet based on corn, soya bean meal and Chinese double-low rapeseed meal.

    PubMed

    Fang, Z F; Peng, J; Liu, Z L; Liu, Y G

    2007-08-01

    This study was conducted to investigate the effect of two distinct enzyme preparations on nutrients' digestibility and growth performance of growing pigs fed diets based on corn, soya bean meal and Chinese double-low rapeseed meal (DLRM). The two enzyme preparations were Enzyme R, a preparation extracted from fermentation of a non-GMO fungus Penicillum funiculosum, developed for multi-grain and multi-animal species; and Enzyme P, a xylanase preparation from Trichoderma longibrachiatum, for pigs fed corn-based diets only. Both enzymes were tested at 0, 0.25 and 0.50 g/kg feed using 70 crossbred male pigs (Large Yorkshire x Landrace) in five dietary treatments and seven replicates in each treatment, for growth period from 27 to 68 kg live weight in 49 days. Results showed that the supplementation of both enzymes (1) increased total-tract digestibility of dietary energy from 77.5% (control) to 81.4% (Enzyme R, p < 0.05) and 81.9% (Enzyme P, p < 0.05); of neutral detergent fibre from 41.0% (control) to 57.8% (Enzyme R, p < 0.05) and 60.0% (Enzyme P, p < 0.05); (2) improved average daily gain from 786 g (control) to 829 g (Enzyme R, p < 0.05) and 846 g (Enzyme P, p < 0.05); and numerical increases in feed intake from 1.96 kg/pig/day (control) to 2.01 (Enzyme R) and 2.00 (p > 0.05) and feed conversion ratio from 2.50 (control) to 2.42 (Enzyme R) and 2.36 (Enzyme P, p < 0.05); (3) there was a dose response but no significant differences were observed in enzyme efficacy between the two enzyme preparations. The present study demonstrated beneficial effects of applying xylanase-based enzymes to improve feeding values of pig diets based on corn, soya bean meal and DLRM.

  16. Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

    PubMed

    Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

    2009-12-11

    Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

  17. EPA's Reanalysis of Key Issues Related to Dioxin Toxicity and Response to NAS Comments (External Review Draft)

    EPA Science Inventory

  1. Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

    PubMed

    Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

    2011-11-01

    There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

  2. Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.

    PubMed

    Lyubenova, Lyudmila; Nehnevajova, Erika; Herzig, Rolf; Schröder, Peter

    2009-07-01

    Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the

  3. Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

    PubMed

    Johnson, Patrick A; Park, Hee Joon; Driscoll, Ashley J

    2011-01-01

    Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

  4. The effects of exogenous hormones on rooting process and the activities of key enzymes of Malus hupehensis stem cuttings.

    PubMed

    Zhang, Wangxiang; Fan, Junjun; Tan, Qianqian; Zhao, Mingming; Zhou, Ting; Cao, Fuliang

    2017-01-01

    Malus hupehensis is an excellent Malus rootstock species, known for its strong adverse-resistance and apomixes. In the present study, stem cuttings of M. hupehensis were treated with three types of exogenous hormones, including indole acetic acid (IAA), naphthalene acetic acid (NAA), or green growth regulator (GGR). The effects and mechanisms of exogenous hormone treatment and antioxidant enzyme activity on adventitious root formation were investigated. The results showed that the apparent morphology of the adventitious root had four stages, including root pre-emergence stage (S0), early stage of root formation (S1), massive root formation stage (S2), and later stage of root formation (S3). The suitable concentrations of the three exogenous hormones, IAA, NAA and GGR, were 100 mg·L-1, 300 mg·L-1, and 300 mg·L-1, respectively. They shortened the rooting time by 25-47.4% and increased the rooting percentages of cuttings by 0.9-1.3 times, compared with that in the control. The dispersion in S0 stage was 3.6 times of that in the S1 stage after exogenous hormone application. The earlier the third critical point (P3) appeared, the shorter the rooting time and the greater the rooting percentage of the cuttings. During rhizogenesis, the activities of three antioxidant enzymes (POD, SOD, and PPO) showed an A-shaped trend. However, peak values of enzyme activity appeared at different points, which were 9 d before the P3, P3, and the fourth critical point (P4), respectively. Exogenous hormone treatment reduced the time to reach the peak value by 18 days, although the peak values of the enzymatic activities did not significantly changed. Our results suggested that exogenous hormone treatment mainly acted during the root pre-emergence stage, accelerated the synthesis of antioxidant enzymes, reduced the rooting time, and consequently promoted root formation. The three kinds of antioxidant enzymes acted on different stages of rooting.

  5. The effects of exogenous hormones on rooting process and the activities of key enzymes of Malus hupehensis stem cuttings

    PubMed Central

    Tan, Qianqian; Zhao, Mingming; Zhou, Ting; Cao, Fuliang

    2017-01-01

    Malus hupehensis is an excellent Malus rootstock species, known for its strong adverse-resistance and apomixes. In the present study, stem cuttings of M. hupehensis were treated with three types of exogenous hormones, including indole acetic acid (IAA), naphthalene acetic acid (NAA), or green growth regulator (GGR). The effects and mechanisms of exogenous hormone treatment and antioxidant enzyme activity on adventitious root formation were investigated. The results showed that the apparent morphology of the adventitious root had four stages, including root pre-emergence stage (S0), early stage of root formation (S1), massive root formation stage (S2), and later stage of root formation (S3). The suitable concentrations of the three exogenous hormones, IAA, NAA and GGR, were 100 mg·L-1, 300 mg·L-1, and 300 mg·L-1, respectively. They shortened the rooting time by 25–47.4% and increased the rooting percentages of cuttings by 0.9–1.3 times, compared with that in the control. The dispersion in S0 stage was 3.6 times of that in the S1 stage after exogenous hormone application. The earlier the third critical point (P3) appeared, the shorter the rooting time and the greater the rooting percentage of the cuttings. During rhizogenesis, the activities of three antioxidant enzymes (POD, SOD, and PPO) showed an A-shaped trend. However, peak values of enzyme activity appeared at different points, which were 9 d before the P3, P3, and the fourth critical point (P4), respectively. Exogenous hormone treatment reduced the time to reach the peak value by 18 days, although the peak values of the enzymatic activities did not significantly changed. Our results suggested that exogenous hormone treatment mainly acted during the root pre-emergence stage, accelerated the synthesis of antioxidant enzymes, reduced the rooting time, and consequently promoted root formation. The three kinds of antioxidant enzymes acted on different stages of rooting. PMID:28231330

  6. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    NASA Astrophysics Data System (ADS)

    Zhou, Xiaoqi; Wang, Shen S. J.; Chen, Chengrong

    2017-12-01

    Forest plantations have been widely used as an effective measure for increasing soil carbon (C), and nitrogen (N) stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA) to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species-enzyme-C/N model to investigate how temperature and tree species influence soil C/N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG), N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP) and phosphorus acquisition enzymes (acid phosphatases). The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01-2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99-2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii) and hoop pine (Araucaria cunninghamii Ait.), increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22-1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native coniferous tree species like hoop pine and

  7. Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

    NASA Astrophysics Data System (ADS)

    Zhang, Zhihua; Qu, Yinbo; Zhang, Xiao; Lin, Jianqiang

    The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h-1 when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.

  8. Canceling effect leads temperature insensitivity of hydrolytic enzymes in soil

    NASA Astrophysics Data System (ADS)

    Razavi, Bahar S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Extracellular enzymes are important for decomposition of many macromolecules abundant in soil such as cellulose, hemicelluloses and proteins (Allison et al., 2010; Chen et al., 2012). The temperature sensitivity of enzymes responsible for organic matter decomposition is the most crucial parameter for prediction of the effects of global warming on carbon cycle. Temperature responses of biological systems are often expressed as a Q10 functions; The Q10 describes how the rate of a chemical reaction changes with a temperature increase for 10 °C The aim of this study was to test how the canceling effect will change with variation in temperature interval, during short-term incubation. We additionally investigated, whether canceling effect occurs in a broad range of concentrations (low to high) and whether it is similar for the set of hydrolytic enzymes within broad range of temperatures. To this end, we performed soil incubation over a temperature range of 0-40°C (with 5°C steps). We determined the activities of three enzymes involved in plant residue decomposition: β-glucosidase and cellobiohydrolase, which are commonly measured as enzymes responsible for degrading cellulose (Chen et al., 2012), and xylanase, which degrades xylooligosaccharides (short xylene chain) in to xylose, thus being responsible for breaking down hemicelluloses (German et al., 2011). Michaelis-Menten kinetics measured at each temperature allowed to calculate Q10 values not only for the whole reaction rates, but specifically for maximal reaction rate (Vmax) and substrate affinity (Km). Subsequently, the canceling effect - simultaneous increase of Vmax and Km with temperature was analyzed within 10 and 5 degree of temperature increase. Three temperature ranges (below 10, between 15 and 25, and above 30 °C) clearly showed non-linear but stepwise increase of temperature sensitivity of all three enzymes and allowed to conclude for predominance of psychrophilic, mesophilic and thermophilic

  9. Iron-mediated soil carbon response to water-table decline in an alpine wetland

    PubMed Central

    Wang, Yiyun; Wang, Hao; He, Jin-Sheng; Feng, Xiaojuan

    2017-01-01

    The tremendous reservoir of soil organic carbon (SOC) in wetlands is being threatened by water-table decline (WTD) globally. However, the SOC response to WTD remains highly uncertain. Here we examine the under-investigated role of iron (Fe) in mediating soil enzyme activity and lignin stabilization in a mesocosm WTD experiment in an alpine wetland. In contrast to the classic ‘enzyme latch’ theory, phenol oxidative activity is mainly controlled by ferrous iron [Fe(II)] and declines with WTD, leading to an accumulation of dissolvable aromatics and a reduced activity of hydrolytic enzyme. Furthermore, using dithionite to remove Fe oxides, we observe a significant increase of Fe-protected lignin phenols in the air-exposed soils. Fe oxidation hence acts as an ‘iron gate’ against the ‘enzyme latch’ in regulating wetland SOC dynamics under oxygen exposure. This newly recognized mechanism may be key to predicting wetland soil carbon storage with intensified WTD in a changing climate. PMID:28649988

  10. Iron-mediated soil carbon response to water-table decline in an alpine wetland

    NASA Astrophysics Data System (ADS)

    Wang, Yiyun; Wang, Hao; He, Jin-Sheng; Feng, Xiaojuan

    2017-06-01

    The tremendous reservoir of soil organic carbon (SOC) in wetlands is being threatened by water-table decline (WTD) globally. However, the SOC response to WTD remains highly uncertain. Here we examine the under-investigated role of iron (Fe) in mediating soil enzyme activity and lignin stabilization in a mesocosm WTD experiment in an alpine wetland. In contrast to the classic `enzyme latch' theory, phenol oxidative activity is mainly controlled by ferrous iron [Fe(II)] and declines with WTD, leading to an accumulation of dissolvable aromatics and a reduced activity of hydrolytic enzyme. Furthermore, using dithionite to remove Fe oxides, we observe a significant increase of Fe-protected lignin phenols in the air-exposed soils. Fe oxidation hence acts as an `iron gate' against the `enzyme latch' in regulating wetland SOC dynamics under oxygen exposure. This newly recognized mechanism may be key to predicting wetland soil carbon storage with intensified WTD in a changing climate.

  11. Optimization of Answer Keys for Script Concordance Testing: Should We Exclude Deviant Panelists, Deviant Responses, or Neither?

    ERIC Educational Resources Information Center

    Gagnon, Robert; Lubarsky, Stuart; Lambert, Carole; Charlin, Bernard

    2011-01-01

    The Script Concordance Test (SCT) uses a panel-based, aggregate scoring method that aims to capture the variability of responses of experienced practitioners to particular clinical situations. The use of this type of scoring method is a key determinant of the tool's discriminatory power, but deviant answers could potentially diminish the…

  12. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    PubMed

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

    PubMed Central

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  14. Proteomic researches for lignocellulose-degrading enzymes: A mini-review.

    PubMed

    Guo, Hongliang; Wang, Xiao-Dong; Lee, Duu-Jong

    2018-05-31

    Protective action of lignin/hemicellulose networks and crystalline structures of embedded cellulose render lignocellulose material resistant to external enzymatic attack. To eliminate this bottleneck, research has been conducted in which advanced proteomic techniques are applied to identify effective commercial hydrolytic enzymes. This mini-review summarizes researches on lignocellulose-degrading enzymes, the mechanisms of the responses of various lignocellulose-degrading strains and microbial communities to various carbon sources and various biomass substrates, post-translational modifications of lignocellulose-degrading enzymes, new lignocellulose-degrading strains, new lignocellulose-degrading enzymes and a new method of secretome analysis. The challenges in the practical use of enzymatic hydrolysis process to realize lignocellulose biorefineries are discussed, along with the prospects for the same. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Understanding Biological Rates and their Temperature Dependence, from Enzymes to Ecosystems

    NASA Astrophysics Data System (ADS)

    Prentice, E.; Arcus, V. L.

    2017-12-01

    Temperature responses over various scales in biological systems follow a similar pattern; negative curvature results in an optimum temperature (Topt) for activity/growth/turnover, with decreases in rates on either side of Topt. Previously this downturn in rates at high temperatures has been attributed to enzyme denaturation, where a failing of the basic driving units of metabolism was used to describe curvature at the enzyme and organism level. However, recent developments in our understanding of the factors governing enzyme rates at different temperatures have guided a new understanding of the responses of biological systems. Enzymes catalyse reactions by driving the substrate through a high energy species, which is tightly bound to the enzyme. Macromolecular rate theory (MMRT) has recently been developed to account for the changes in the system brought about by this tight binding, specifically the change in the physical parameter heat capacity (ΔCǂp), and the effect this has on the temperature dependence of enzyme reactions. A negative ΔCǂp imparts the signature negative curvature to rates in the absence of denaturation, and finds that Topt, ΔCǂp and curvature are all correlated, placing constraints on biological systems. The simplest of cells comprise thousands of enzymatically catalysed reactions, functioning in series and in parallel in metabolic pathways to determine the overall growth rate of an organism. Intuitively, the temperature effects of enzymes play a role in determining the overall temperature dependence of an organism, in tandem with cellular level regulatory responses. However, the effect of individual Topt values and curvature on overall pathway behaviour is less apparent. Here, this is investigated in the context of MMRT through the in vitro characterisation of a six-step metabolic pathway to understand the steps in isolation and functioning in series. Pathway behaviour is found to be approximately an average of the properties of the

  16. Enzyme-based logic gates and circuits-analytical applications and interfacing with electronics.

    PubMed

    Katz, Evgeny; Poghossian, Arshak; Schöning, Michael J

    2017-01-01

    The paper is an overview of enzyme-based logic gates and their short circuits, with specific examples of Boolean AND and OR gates, and concatenated logic gates composed of multi-step enzyme-biocatalyzed reactions. Noise formation in the biocatalytic reactions and its decrease by adding a "filter" system, converting convex to sigmoid response function, are discussed. Despite the fact that the enzyme-based logic gates are primarily considered as components of future biomolecular computing systems, their biosensing applications are promising for immediate practical use. Analytical use of the enzyme logic systems in biomedical and forensic applications is discussed and exemplified with the logic analysis of biomarkers of various injuries, e.g., liver injury, and with analysis of biomarkers characteristic of different ethnicity found in blood samples on a crime scene. Interfacing of enzyme logic systems with modified electrodes and semiconductor devices is discussed, giving particular attention to the interfaces functionalized with signal-responsive materials. Future perspectives in the design of the biomolecular logic systems and their applications are discussed in the conclusion. Graphical Abstract Various applications and signal-transduction methods are reviewed for enzyme-based logic systems.

  17. Maori responsiveness in health and medical research: key issues for researchers (part 1).

    PubMed

    Sporle, Andrew; Koea, Jonathan

    2004-08-06

    Application for contestable government-research funding and ethical approval requires researchers to outline how their intended research project contributes to Maori development or advancement. When formulating their research proposals, the key issues for researchers are research utility, defining Maori, informed consent, confidentiality, issues with human tissues and genetic material, participant remuneration and recognition (koha), intellectual property, and involvement of local Maori health or social services. The most common Maori responsiveness issues in research applications can be readily approached by researchers who address straightforward methodological concerns, by working through precedents established by peers and colleagues, as well as by working with end-users of their research.

  18. Key Enzymes Enabling the Growth of Arthrobacter sp. Strain JBH1 with Nitroglycerin as the Sole Source of Carbon and Nitrogen

    PubMed Central

    Husserl, Johana; Hughes, Joseph B.

    2012-01-01

    Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG. PMID:22427495

  19. Key enzymes enabling the growth of Arthrobacter sp. strain JBH1 with nitroglycerin as the sole source of carbon and nitrogen.

    PubMed

    Husserl, Johana; Hughes, Joseph B; Spain, Jim C

    2012-05-01

    Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG.

  20. Cooperativity in Monomeric Enzymes with Single Ligand-Binding Sites

    PubMed Central

    Porter, Carol M.

    2011-01-01

    Cooperativity is widespread in biology. It empowers a variety of regulatory mechanisms and impacts both the kinetic and thermodynamic properties of macromolecular systems. Traditionally, cooperativity is viewed as requiring the participation of multiple, spatially distinct binding sites that communicate via ligand-induced structural rearrangements; however, cooperativity requires neither multiple ligand binding events nor multimeric assemblies. An underappreciated manifestation of cooperativity has been observed in the non-Michaelis-Menten kinetic response of certain monomeric enzymes that possess only a single ligand-binding site. In this review, we present an overview of kinetic cooperativity in monomeric enzymes. We discuss the primary mechanisms postulated to give rise to monomeric cooperativity and highlight modern experimental methods that could offer new insights into the nature of this phenomenon. We conclude with an updated list of single subunit enzymes that are suspected of displaying cooperativity, and a discussion of the biological significance of this unique kinetic response. PMID:22137502

  1. Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

    PubMed Central

    Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W. H.; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2010-01-01

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, 163DVD165 and 221FWGWGREDDE230, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp228 acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics. PMID:20843813

  2. Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: Implication in designing of novel anticancer therapeutic strategies

    PubMed Central

    Yadav, Saveg; Pandey, Shrish Kumar; Singh, Vinay Kumar; Goel, Yugal; Kumar, Ajay

    2017-01-01

    Altered metabolism is an emerging hallmark of cancer, as malignant cells display a mammoth up-regulation of enzymes responsible for steering their bioenergetic and biosynthetic machinery. Thus, the recent anticancer therapeutic strategies focus on the targeting of metabolic enzymes, which has led to the identification of specific metabolic inhibitors. One of such inhibitors is 3-bromopyruvate (3-BP), with broad spectrum of anticancer activity due to its ability to inhibit multiple metabolic enzymes. However, the molecular characterization of its binding to the wide spectrum of target enzymes remains largely elusive. Therefore, in the present study we undertook in silico investigations to decipher the molecular nature of the docking of 3-BP with key target enzymes of glycolysis and TCA cycle by PatchDock and YASARA docking tools. Additionally, derivatives of 3-BP, dibromopyruvate (DBPA) and propionic acid (PA), with reported biological activity, were also investigated for docking to important target metabolic enzymes of 3-BP, in order to predict their therapeutic efficacy versus that of 3-BP. A comparison of the docking scores with respect to 3-BP indicated that both of these derivatives display a better binding strength to metabolic enzymes. Further, analysis of the drug likeness of 3-BP, DBPA and PA by Lipinski filter, admetSAR and FAF Drug3 indicated that all of these agents showed desirable drug-like criteria. The outcome of this investigation sheds light on the molecular characteristics of the binding of 3-BP and its derivatives with metabolic enzymes and thus may significantly contribute in designing and optimizing therapeutic strategies against cancer by using these agents. PMID:28463978

  3. Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: Implication in designing of novel anticancer therapeutic strategies.

    PubMed

    Yadav, Saveg; Pandey, Shrish Kumar; Singh, Vinay Kumar; Goel, Yugal; Kumar, Ajay; Singh, Sukh Mahendra

    2017-01-01

    Altered metabolism is an emerging hallmark of cancer, as malignant cells display a mammoth up-regulation of enzymes responsible for steering their bioenergetic and biosynthetic machinery. Thus, the recent anticancer therapeutic strategies focus on the targeting of metabolic enzymes, which has led to the identification of specific metabolic inhibitors. One of such inhibitors is 3-bromopyruvate (3-BP), with broad spectrum of anticancer activity due to its ability to inhibit multiple metabolic enzymes. However, the molecular characterization of its binding to the wide spectrum of target enzymes remains largely elusive. Therefore, in the present study we undertook in silico investigations to decipher the molecular nature of the docking of 3-BP with key target enzymes of glycolysis and TCA cycle by PatchDock and YASARA docking tools. Additionally, derivatives of 3-BP, dibromopyruvate (DBPA) and propionic acid (PA), with reported biological activity, were also investigated for docking to important target metabolic enzymes of 3-BP, in order to predict their therapeutic efficacy versus that of 3-BP. A comparison of the docking scores with respect to 3-BP indicated that both of these derivatives display a better binding strength to metabolic enzymes. Further, analysis of the drug likeness of 3-BP, DBPA and PA by Lipinski filter, admetSAR and FAF Drug3 indicated that all of these agents showed desirable drug-like criteria. The outcome of this investigation sheds light on the molecular characteristics of the binding of 3-BP and its derivatives with metabolic enzymes and thus may significantly contribute in designing and optimizing therapeutic strategies against cancer by using these agents.

  4. Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.

    PubMed

    Niwa, Ryusuke; Niwa, Yuko S

    2014-01-01

    Steroid hormones are responsible for the coordinated regulation of many aspects of biological processes in multicellular organisms. Since the last century, many studies have identified and characterized steroidogenic enzymes in vertebrates, including mammals. However, much less is known about invertebrate steroidogenic enzymes. In the last 15 years, a number of steroidogenic enzymes and their functions have been characterized in ecdysozoan animals, especially in the fruit fly Drosophila melanogaster. In this review, we summarize the latest knowledge of enzymes crucial for synthesizing ecdysteroids, the principal insect steroid hormones. We also discuss the functional conservation and diversity of ecdysteroidogenic enzymes in other insects and even non-insect species, such as nematodes, vertebrates, and lower eukaryotes.

  5. Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora.

    PubMed

    Norman-Setterblad, C; Vidal, S; Palva, E T

    2000-04-01

    We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

  6. An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening.

    PubMed

    Vilela, Adriana Ferreira Lopes; Seidl, Cláudia; Lima, Juliana Maria; Cardoso, Carmen Lúcia

    2018-05-15

    Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H] + m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC 50 values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results. Copyright © 2018. Published by Elsevier Inc.

  7. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Impaired small-bowel barrier integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory shock.

    PubMed

    Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W

    2012-08-01

    In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress

  9. IMPAIRED SMALL BOWEL BARRIER INTEGRITY IN THE PRESENCE OF LUMENAL PANCREATIC DIGESTIVE ENZYMES LEADS TO CIRCULATORY SHOCK

    PubMed Central

    Kistler, Erik B.; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W.

    2012-01-01

    In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These

  10. Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia.

    PubMed

    Swords, Ronan T; Kelly, Kevin R; Smith, Peter G; Garnsey, James J; Mahalingam, Devalingam; Medina, Ernest; Oberheu, Kelli; Padmanabhan, Swaminathan; O'Dwyer, Michael; Nawrocki, Steffan T; Giles, Francis J; Carew, Jennifer S

    2010-05-06

    NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

  11. Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

    PubMed

    Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

    2016-09-15

    Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

  12. 'The ones that turn up are the ones that are responsible': Key stakeholders perspectives on liquor accords.

    PubMed

    Curtis, Ashlee; Miller, Peter; Droste, Nicolas; McFarlane, Emma; Martino, Florentine; Palmer, Darren

    2016-05-01

    Liquor accords were introduced as an intervention to reduce alcohol-related harm in and around licensed venues. There have been very few evaluations of the accords, made all the more difficult given the multitude of measures that are often implemented under their banner. This study provides perspectives on the effectiveness of the liquor accords from key stakeholders who were involved in the strategy. In-depth interviews were conducted with 97 key stakeholders as part of a larger study, of which 46 spoke about the effectiveness of liquor accords. Responses were analysed using thematic analysis. Stakeholders reported the greatest benefit of liquor accords to be their ability to improve communication. Many stakeholders recognised the need for mandatory attendance and discussed whether the accords are a waste of time of resources. Stakeholders did not generally view liquor accords as effective means of reducing alcohol-related harm. There was a lack of positive feedback about liquor accords provided by stakeholders, indicating a clear need to better understand the role of liquor accords, and what they aim to achieve. Responsive regulation theory suggests that the dual roles of communication and intervention are confused, leading to some of the inherent problems with accords. The role and aims of liquor accords need to be clearly defined. The findings suggest that separating the communication and regulatory functions from accords will lead to a clearer role for accords, and interventions and regulation might be better placed in the hands of regulators and enforcement. [Curtis A, Miller P, Droste N, McFarlane E, Martino F, Palmer D. 'The ones that turn up are the ones that are responsible': Key stakeholders perspectives on liquor accords. Drug Alcohol Rev 2016;35:273-279]. © 2016 Australasian Professional Society on Alcohol and other Drugs.

  13. Enzyme Recruitment and Its Role in Metabolic Expansion

    PubMed Central

    2015-01-01

    Although more than 109 years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology. PMID:24483367

  14. A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

    PubMed Central

    Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff S.; Tang, Christoph M.

    2013-01-01

    Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. PMID:22296581

  15. Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Ma, Yuexin; Sun, Feixue; Zhang, Congyao; Bao, Pengyun; Cao, Shuqing; Zhang, Meiyan

    2014-12-01

    A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0 (control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control ( P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet ( P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets ( P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

  16. Enzyme dynamics in paddy soils of the rice district (NE Italy) under different cropping patterns

    NASA Astrophysics Data System (ADS)

    Bini, Claudio; Nadimi-Goki, Mandana; Kato, Yoichi; Fornasier, Flavio; Wahsha, Mohammad; Spiandorello, Massimo

    2014-05-01

    The recent widespread interest on soil enzymes is due to the need to develop sensitive indicators of soil quality that reflect the effects of land management on soil and assist land managers in promoting long-term sustainability of terrestrial ecosystems. The activities of six important enzymes involved in C, N, P, and S cycling were investigated in a paddy soil from the Veneto region, Italy, in four different rotation systems (rice-rice-rice: R-R-R; soya-rice-rice: S-R-R; fallow-rice: F-R; pea-soya-rice: P-S-R) with three replications in April (after field preparation, field moist condition), June (after seedling, waterlogged soil condition), August (after tillering stage of rice, waterlogged soil condition) and October (after rice harvesting, drained soil condition) over the 2012 growing season. Our results demonstrated that enzyme activities varied with rotation systems and growth stages in paddy soil. Compared with field moist soil, drained soil condition resulted in a significant increase (P < 0.05) of β-glucosidase, arylsulfatase, alkaline and acid phosphatases, leucine aminopeptidase (except of fallow-rice), and chitinase activities in all rotations, while compared with drained soil, early waterlogging (in month of June) significantly decreased (P moist soil> late waterlogged>early waterlogged. There was an inhibitory effect of waterlogging (except P-S-R rotation) for both alkaline and acid phosphatases due to high pH and redox conditions. However, the response of enzymes to waterlogging differed with the chemical species and the cropping pattern. The best rotation system for chitinase, leucine aminopeptidase and β-glucosidase activity (C and N cycles) proved R-R-R, while for arylsulfatase, alkaline and acid phosphatases (P and S cycles) it was the S-R-R. Key Words: enzyme activity, paddy soil, Crop Rotation System, Italy __ Corresponding Author: Mandana Nadimi-Goki, Tel.: +39 3891356251 E-mail address: mandy.nadimi@gmail.com

  17. Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome.

    PubMed

    Kattah, Michael G; Malynn, Barbara A; Ma, Averil

    2017-11-10

    Ubiquitin and ubiquitin-modifying enzymes play critical roles in a wide variety of intracellular signaling pathways. Inflammatory signaling cascades downstream of TNF, TLR agonists, antigen receptor cross-linking, and cytokine receptors, all rely on ubiquitination events to direct subsequent immune responses. In the past several years, inflammasome activation and subsequent signal transduction have emerged as an excellent example of how ubiquitin signals control inflammatory responses. Inflammasomes are multiprotein signaling complexes that ultimately lead to caspase activation and release of the interleukin-1 (IL-1) family members, IL-1β and IL-18. Inflammasome activation is critical for the host's defense against pathogens, but dysregulation of inflammasomes may contribute to the pathogenesis of multiple diseases. Ultimately, understanding how various ubiquitin interacting proteins control inflammatory signaling cascades could provide new pathways for therapeutic intervention. Here we review specific ubiquitin-modifying enzymes and ubiquitination events that orchestrate inflammatory responses, with an emphasis on the NLRP3 inflammasome. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    PubMed

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  19. Functional and transcriptomic analysis of the key unfolded protein response transcription factor HacA in Aspergillus oryzae.

    PubMed

    Zhou, Bin; Xie, Jingyi; Liu, Xiaokai; Wang, Bin; Pan, Li

    2016-11-15

    HacA is a conserved basic leucine zipper transcription factor that serves as the master transcriptional regulator in the unfolded protein response (UPR). To comprehensively evaluate the role of HacA in Aspergillus oryzae, a homokaryotic hacA disruption mutant (HacA-DE) and a strain that expressed a constitutively active form of HacA (HacA-CA) were successfully generated, and transcriptome analyses of these mutants were performed. Growth and phenotypic profiles demonstrated that hyphal growth and sporulation were impaired in the HacA-DE and HacA-CA strains that were grown on complete and minimal media, and the growth impairment was more pronounced for the HacA-CA strain. Compared with a wild-type (WT) strain, the transcriptome results indicated that differentially expressed genes in these mutants mainly fell into four categories: the protein secretory pathway, amino acid metabolism, lipid metabolism, and carbohydrate metabolism. Furthermore, we identified 80 and 36 genes of the secretory pathway whose expression significantly differed in the HacA-CA strain (compared with the WT and HacA-DE strains) and HacA-DE strain (compared with the WT strain), respectively, which mostly belonged to protein folding/UPR, glycosylation, and vesicle transport processes. Both the HacA-CA and HacA-DE strains exhibited reduced expression of extracellular enzymes, especially amylolytic enzymes, which resulted from the activation of the repression under secretion stress mechanism in response to endoplasmic reticulum stress. Collectively, our results suggest that the function of HacA is important not only for UPR induction, but also for growth and fungal physiology, as it serves to reduce secretion stress in A. oryzae. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    PubMed

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

  1. Phospholipid-sepiolite biomimetic interfaces for the immobilization of enzymes.

    PubMed

    Wicklein, Bernd; Darder, Margarita; Aranda, Pilar; Ruiz-Hitzky, Eduardo

    2011-11-01

    Biomimetic interfaces based on phosphatidylcholine (PC) assembled to the natural silicate sepiolite were prepared for the stable immobilization of the urease and cholesterol oxidase enzymes. This is an important issue in practical advanced applications such as biocatalysis or biosensing. The supported lipid bilayer (BL-PC), prepared from PC adsorption, was used for immobilization of enzymes and the resulting biomimetic systems were compared to several other supported layers including a lipid monolayer (ML-PC), a mixed phosphatidylcholine/octyl-galactoside layer (PC-OGal), a cetyltrimethylammonium monolayer (CTA), and also to the bare sepiolite surface. Interfacial characteristics of these layers were investigated with a focus on layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. Cytoplasmic urease and membrane-bound cholesterol oxidase, which served as model enzymes, were immobilized on the different PC-based hybrid materials to probe their biomimetic character. Enzymatic activity was assessed by cyclic voltammetry and UV-vis spectrophotometry. The resulting enzyme/bio-organoclay hybrids were applied as active phase of a voltammetric urea biosensor and cholesterol bioreactor, respectively. Urease supported on sepiolite/BL-PC proved to maintain its enzymatic activity over several months while immobilized cholesterol oxidase demonstrated high reusability as biocatalyst. The results emphasize the good preservation of bioactivity due to the accommodation of the enzymatic system within the biomimetic lipid interface on sepiolite.

  2. Probing collagen-enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep

    PubMed Central

    Zareian, Ramin; Church, Kelli P.; Saeidi, Nima; Flynn, Brendan P.; Beale, John W.; Ruberti, Jeffrey W.

    2012-01-01

    Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially-loaded strips of bovine corneal tissue (0.1, 0.25 or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at ~5% strain) reduces the maximum cutting-rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective “cutoff” occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is non-linear (initially-steep between 4.5 and 6.5% then flattens out from 6.5–9.5%). The high sensitivity to strain at low strain implies that even lightly-loaded collagenous tissue may exhibit significant strain-protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme

  3. TISSUE ENZYME RESPONSE TO COLD AND TO HYPERPHAGIA IN THE RAT,

    DTIC Science & Technology

    activated glutaminase were increased. In animals with a comparable hyperphagia due to bilateral ablation of the ventromedial region of the hypothalamus...concluded that changes of enzyme activities in cold-exposed rats are not simply an adaptation to the increased nutrient flow consequent upon the hyperphagia induced. (Author)

  4. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

    PubMed Central

    Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

    2005-01-01

    4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

  5. [Human drug metabolizing enzymes. II. Conjugation enzymes].

    PubMed

    Vereczkey, L; Jemnitz, K; Gregus, Z

    1998-09-01

    In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

  6. Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

    PubMed

    Habig, Michael; Blechschmidt, Anke; Dressler, Sigmar; Hess, Barbara; Patel, Viral; Billich, Andreas; Ostermeier, Christian; Beer, David; Klumpp, Martin

    2009-07-01

    High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

  7. Genetic polymorphisms of enzyme proteins and transporters related to methotrexate response and pharmacokinetics in a Japanese population.

    PubMed

    Hashiguchi, Masayuki; Shimizu, Mikiko; Hakamata, Jun; Tsuru, Tomomi; Tanaka, Takanori; Suzaki, Midori; Miyawaki, Kumika; Chiyoda, Takeshi; Takeuchi, Osamu; Hiratsuka, Jiro; Irie, Shin; Maruyama, Junya; Mochizuki, Mayumi

    2016-01-01

    Methotrexate (MTX) is currently the anchor drug widely used worldwide in the treatment of rheumatoid arthritis (RA). However, the therapeutic response to MTX has been shown to vary widely among individuals, genders and ethnic groups. The reason for this has been not clarified but it is considered to be partially due to several mechanisms in the cellular pathway of MTX including single-nucleotide polymorphisms (SNPs). The purpose of this study was to investigate the allelic frequencies in different ethnic and/or population groups in the 10 polymorphisms of enzyme proteins and transporters related to the MTX response and pharmacokinetics including MTHFR, TYMS, RFC1, FPGS, GGH, ABCB1, ABCC2 and ABCG2 in unrelated healthy Japanese adults and patients with RA. Ten polymorphisms, methylenetetrahydrofolate reductase (MTHFR) 1298, thymidylate synthase (TYMS) 3'-UTR, reduced folate carrier 1 (RFC1) 80 and-43, folypolyglutamyl synthase (FPGS) 1994, γ-glutamyl hydrolase (GGH) 452 and-401, the ABC transporters (ABCB1 3435, ABCC2 IVS23 + 56, ABCG2 914) of enzyme proteins and transporters related to MTX response and pharmacokinetics in 299 unrelated healthy Japanese adults and 159 Japanese patients with RA were investigated to clarify their contributions to individual variations in response and safety to MTX and establish personalized MTX therapy. SNPs were evaluated using real-time polymerase chain reaction (PCR). Comparison of allelic frequencies in our study with other ethnic/population groups of healthy adults and RA patients showed significant differences in 10 polymorphisms among healthy adults and 7 among RA patients. Allelic frequencies of MTHFR 1298 C, FPGS 1994A and ABCB1 3435 T were lower in Japanese than in Caucasian populations and those of ABCC2 IVS23 + 56 C and ABCG2 914A were higher in Japanese than in Caucasian/European populations in both healthy adults and RA patients. Allelic frequencies of MTHFR 1298 C, GGH-401 T, ABCB1 3435 T, and ABCG2 914A

  8. Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

    PubMed

    Zhdanov, Alexander V; Waters, Alicia H C; Golubeva, Anna V; Dmitriev, Ruslan I; Papkovsky, Dmitri B

    2014-01-01

    Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment. © 2013.

  9. Rational Design of Thermally Stable Novel Biocatalytic Nanomaterials: Enzyme Stability in Restricted Spatial Dimensions

    NASA Astrophysics Data System (ADS)

    Mudhivarthi, Vamsi K.

    Enzyme stability is of intense interest in bio-materials science as biocatalysts, and as sensing platforms. This is essentially because the unique properties of DNA, RNA, PAA can be coupled with the interesting and novel properties of proteins to produce systems with unprecedented control over their properties. In this article, the very first examples of enzyme/NA/inorganic hybrid nanomaterials and enzyme-Polyacrylic acid conjugates will be presented. The basic principles of design, synthesis and control of properties of these hybrid materials will be presented first, and this will be followed by a discussion of selected examples from our recent research findings. Data show that key properties of biological catalysts are improved by the inorganic framework especially when the catalyst is co-embedded with DNA. Several examples of such studies with various enzymes and proteins, including horseradish peroxidase (HRP), glucose oxidase (GO), cytochrome c (Cyt c), met-hemoglobin (Hb) and met-myoglobin (Mb) will be discussed. Additionally, key insights obtained by the standard methods of materials science including XRD, SEM and TEM as well as biochemical, calorimetric and spectroscopic methods will be discussed. Furthermore, improved structure and enhanced activities of the biocatalysts in specific cases will be demonstrated along with the potential stabilization mechanisms. Our hypothesis is that nucleic acids provide an excellent control over the enzyme-solid interactions as well as rational assembly of nanomaterials. These novel nanobiohybrid materials may aid in engineering more effective synthetic materials for gene-delivery, RNA-delivery and drug delivery applications.

  10. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    PubMed

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

    2016-01-01

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

  11. Structure and Function of Viral Deubiquitinating Enzymes.

    PubMed

    Bailey-Elkin, Ben A; Knaap, Robert C M; Kikkert, Marjolein; Mark, Brian L

    2017-11-10

    Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Enzyme-based fiber optic sensors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kulp, T.J.; Camins, I.; Angel, S.M.

    Fiber optic chemical sensors capable of detecting glucose and penicillin were developed. Each consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme and a pH-sensitive fluorescent dye (fluorescein). A signal is produced when the enzyme catalyzes the conversion of the analyte (glucose or penicillin) into a product (gluconic or penicilloic acid, respectively) that lowers the microenvironmental pH of the membrane and consequently, lowers the fluorescence intensity of the dye. Each sensor is capable of responding to analyte concentrations in the range of approx.0.1 to 100 mM. Themore » penicillin optrode response time is 40 to 60 s while that for glucose is approx.5 to 12 min. 7 figs.« less

  13. Comparison of Soluble and Immobilised Enzymes

    ERIC Educational Resources Information Center

    Wiseman, Alan

    2003-01-01

    This short article was written in response to a proposed practical featured in the Spring 2002 issue of the "Journal of Biological Education." Beaumont, Cotterill and Williams described a system representing a useful way by which the deleterious effects of free radical attack on enzymes can be demonstrated to undergraduate bioscience students,…

  14. Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging.

    PubMed

    Chyan, Wen; Raines, Ronald T

    2018-06-20

    Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chemical biology. Those probes that respond to enzymatic activity illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, we review recent advances in enzyme-activated fluorogenic probes for biological imaging. We organize our survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside of therapeutic, diagnostic, and theranostic opportunities.

  15. Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes

    NASA Astrophysics Data System (ADS)

    Sydor, Paulina K.; Barry, Sarah M.; Odulate, Olanipekun M.; Barona-Gomez, Francisco; Haynes, Stuart W.; Corre, Christophe; Song, Lijiang; Challis, Gregory L.

    2011-05-01

    Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.

  16. Deactivating Chemical Agents Using Enzyme-Coated Nanofibers Formed by Electrospinning

    DTIC Science & Technology

    2016-01-01

    7.3mM/mg). Key words Coaxial electrospinning, DFPase, Enzyme, chemical warfare , nanofiber, decontamination . Introduction Chemical warfare ...Krile, R.; Nishioka, M.; Taylor, M.; Riggs, K.; Stone, H. Decontamination of Toxic Industrial Chemicals and Chemical Warfare Agents On Building...298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 MATS COATINGS ELECTROSPINNING CHEMICAL WARFARE

  17. Changes in body-weight, composition and hepatic enzyme activities in response to dietary methionine, betaine and choline levels in growing chicks.

    PubMed

    Saunderson, C L; Mackinlay, J

    1990-03-01

    The experiments described here were set up (a) to investigate the effect of age and (b) to investigate the effect of giving five diets which varied in methionine and choline or betaine contents on some of the enzymes that metabolize these nutrients in chick liver. Growth and carcass composition of the chicks fed on the different diets were also examined. There was no obvious relationship between age and enzyme activity in young chicks. Only a diet low in methionine (but not one low in choline) showed a significant decrease in growth and a change in carcass composition. The effects of diet on enzyme activity were complex. Choline oxidase (EC 1.1.3.17) activity was affected by the level of choline in the diet, being high when choline was present at high levels, especially when methionine was limiting. 5-Methyltetrahydrofolate homocysteine methyltransferase (EC 2.1.1.3) had a high activity in the livers of chicks fed on a conventional diet compared with those given semi-purified diets. Other enzymes showed minor changes in response to the diet. The diet low in methionine showed a lower activity of cystathionine beta-synthase (EC 4.2.1.22) and slightly higher activities of methionine adenosyltransferase (EC 2.5.1.6) and betaine-homocysteine methyltransferase (EC 2.1.1.5; compared with other diets), suggesting that this diet encouraged re-methylation of homocysteine at the expense of trans-sulphuration to cystathionine. The findings obtained in these studies form a useful basis for further investigation of the metabolic interrelationships between methionine and related nutrients.

  18. Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

    PubMed

    Kürten, Charlotte; Syrén, Per-Olof

    2016-01-16

    Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

  19. Identification and characterization of gamma-glutamylamine cyclotransferase, an enzyme responsible for gamma-glutamyl-epsilon-lysine catabolism.

    PubMed

    Oakley, Aaron J; Coggan, Marjorie; Board, Philip G

    2010-03-26

    Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

  20. Identification and Characterization of γ-Glutamylamine Cyclotransferase, an Enzyme Responsible for γ-Glutamyl-ϵ-lysine Catabolism*

    PubMed Central

    Oakley, Aaron J.; Coggan, Marjorie; Board, Philip G.

    2010-01-01

    γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu82). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity. PMID:20110353

  1. [Fabrication and evaluation of the enzyme immunosensor for rapid detection of Vibrio parahaemolyticus based on chitosan-SiO2 hybrid membrane].

    PubMed

    Zhao, Guang-Ying; Ma, Chao; Li, Jian-Rong

    2010-01-01

    To improve the key technology of immunesensors in immobilizing bio-sensitive element and keeping its bioactivity, an enzyme immunosensor based on chitosan-SiO(2) (CS-Sio(2)) hybrid membrane was fabricated. To estimate the new immunosensor Vibrio parahaemolyticus which was the main pathogens of aquatic products. A CS-SiO(2) hybrid membrane was prepared using sol-gel method. The enzyme immunosensor was fabricated by coating the membrane and horseradish peroxidase labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) on the surface of four-channel screen-printed carbon electrode. The immunosensor was characterized by cyclic voltammetry. Vibrio parahaemolyticus could be detected according to the decrease percentage (DP) of peak current before and after immune response, while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction. Seven kinds of bacteria, like Vibrio alginolyticus, were selected for specific experiments. By studying the infrared spectrum of three kinds of films, the CS-SiO(2) hybrid membrane was prepared and HRP-anti-VP was fixed in the hybrid membrane. Under the optimum conditions of immunoreaction and electrochemical detection, the DP of peak current before and after immune response showed a linear relation with lgC in the range of 10(4) - 10(9) cfu/ml, while the linear regression equation was: DP = 6.5 lgC-3.319, the correlation coefficient was 0.9958 and the detection limit was 6.9 x 10(3) cfu/ml (S/N = 3). The immunosensor possessed acceptable specificity, reproducibility (RSD < 6%), stability (the amperometric response was 95% of the initial response after a week) and accuracy (96.7% of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003). The enzyme immunosensor based on CS-SiO(2) hybrid membrane gave a good performance in rapid detection of Vibrio parahaemolyticus.

  2. Extracellular enzyme kinetics scale with resource availability

    USGS Publications Warehouse

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

    2014-01-01

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

  3. Assessment of mercaptopurine (6MP) metabolites and 6MP metabolic key-enzymes in childhood acute lymphoblastic leukemia.

    PubMed

    Wojtuszkiewicz, Anna; Barcelos, Ana; Dubbelman, Boas; De Abreu, Ronney; Brouwer, Connie; Bökkerink, Jos P; de Haas, Valerie; de Groot-Kruseman, Hester; Jansen, Gerrit; Kaspers, Gertjan L; Cloos, Jacqueline; Peters, G J

    2014-01-01

    Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MP's sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N=236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5'-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP--TGN and meTIN--showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions.

  4. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    PubMed

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  5. Maize homologs of HCT, a key enzyme in lignin biosynthesis, bind the NLR Rp1 proteins to modulate the defense response

    USDA-ARS?s Scientific Manuscript database

    In plants, most disease resistance (R) genes encode nucleotide binding leucine-rich-repeat 42 (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) 43 upon pathogen recognition. The maize NLR protein Rp1-D21 derives from an intragenic 44 recombination between...

  6. Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp.

    PubMed

    Kaushik, Neelima; Nadella, Tejaswi; Rao, P Srinivasa

    2015-11-01

    This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6- to 20-min pressure-hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain. Exploration of additional hurdles such as pH, TSS, and temperature for enzyme inactivation during high pressure processing of fruits is useful from industrial point of view, as these parameters play key role in preservation process design. © 2015 Institute of Food Technologists®

  7. Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water.

    PubMed

    Fleeker, J

    1987-01-01

    Two solid-phase enzyme immunoassays were developed to measure 2,4-dichlorophenoxyacetic acid (2,4-D), using 2 sets of structurally distinct immunogens and enzyme ligands. The 2,4-D analog, 2-methyl-4-chlorophenoxyacetic acid (MCPA), gave a similar response with both methods, whereas other phenoxy herbicides cross-reacted differently. In method A, the aromatic moiety of 2,4-D was distal from the carrier protein and labeled enzyme, whereas in method B, the acetic acid portion of the herbicide was distal. The use of both methods to screen for this herbicide in ground water and municipal and river water reduced the number of false-positive responses. Water sources having a low background response could be monitored with either method alone. When a concentration step, with disposable C18 extraction columns, was used, the limit of sensitivity was 5 micrograms/L. Method A was the more sensitive of the 2 methods with a limit of detection of 10 micrograms/L without the concentration step.

  8. A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

    PubMed

    Böhnke, Stefanie; Perner, Mirjam

    2015-03-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

  9. Histochemical location of key enzyme activities involved in receptivity and self-incompatibility in the olive tree (Olea europaea L.).

    PubMed

    Serrano, Irene; Olmedilla, Adela

    2012-12-01

    Stigma-surface and style enzymes are important for pollen reception, selection and germination. This report deals with the histochemical location of the activity of four basic types of enzyme involved in these processes in the olive (Olea europaea L.). The detection of peroxidase, esterase and acid-phosphatase activities at the surface of the stigma provided evidence of early receptivity in olive pistils. The stigma maintained its receptivity until the arrival of pollen. Acid-phosphatase activity appeared in the style at the moment of anthesis and continued until the fertilization of the ovule. RNase activity was detected in the extracellular matrix of the styles of flowers just before pollination and became especially evident in pistils after self-pollination. This activity gradually decreased until it practically disappeared in more advanced stages. RNase activity was also detected in pollen tubes growing in pollinated pistils and appeared after in vitro germination in the presence of self-incompatible pistils. These findings suggest that RNases may well be involved in intraspecific pollen rejection in olive flowers. To the best of our knowledge this is the first time that evidence of enzyme activity in stigma receptivity and pollen selection has been described in this species. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

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

    PubMed

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

    2010-06-01

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

  12. Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

    PubMed

    Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

    2018-01-29

    Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

  13. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  14. Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression.

    PubMed

    Hansen, Truls Wergeland; Folkvord, Arild; Grøtan, Espen; Sæle, Øystein

    2013-03-01

    A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Enzyme actuated bioresponsive hydrogels

    NASA Astrophysics Data System (ADS)

    Wilson, Andrew Nolan

    Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. Conferred with the ability to respond to specific biologically derived stimuli, the design challenge is in effectively linking the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range and limit of detection. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide the sensing, transduction, and the actuation response of hydrogels. To investigate the dynamics of these materials, model systems may be used which seek to interrogate the system dynamics by uni-variable experimentation and limit confounding phenomena such as: polymer-solute interactions, polymer swelling dynamics and biomolecular reaction-diffusion concerns. To this end, a model system, alpha-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel discs were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a

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

    PubMed

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

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

  17. Enzyme-responsive nanocomposites for wound infection prophylaxis in burn management: in vitro evaluation of their compatibility with healing processes

    PubMed Central

    Grützner, Verena; Unger, Ronald E; Baier, Grit; Choritz, Lars; Freese, Christian; Böse, Thomas; Landfester, Katharina; Kirkpatrick, C James

    2015-01-01

    Responsive, theranostic nanosystems, capable of both signaling and treating wound infections, is a sophisticated approach to reduce the most common and potentially traumatizing side effects of burn wound treatment: slowed wound healing due to prophylactic anti-infective drug exposure as well as frequent painful dressing changes. Antimicrobials as well as dye molecules have been incorporated into biodegradable nanosystems that release their content only in the presence of pathogens. Following nanocarrier degradation by bacterial enzymes, any infection will thus emit a visible signal and be effectively treated at its source. In this study, we investigated the effect of fluorescent-labeled hyaluronan nanocapsules containing polyhexanide biguanide and poly-L-lactic acid nanoparticles loaded with octenidine on primary human dermal microvascular endothelial cells, which play a major role in cutaneous wound healing. Microscopic and flow cytometric analysis indicated a time-dependent uptake of both the nanocapsules and the nanoparticles. However, enzyme immunoassays showed no significant influence on the expression of pro-inflammatory cell adhesion molecules and cytokines by the endothelial cells. Under angiogenic-stimulating conditions, the potential to form capillary-like structures in co-culture with dermal fibroblasts was not inhibited. Furthermore, cytotoxicity studies (the MTS and crystal violet assay) after short- and long-term exposure to the materials demonstrated that both systems exhibited less toxicity than solutions of the antiseptic agents alone in comparable concentrations. The results indicate that responsive antimicrobial nanocomposites could be used as an advanced drug delivery system and a promising addition to current best practice wound infection prophylaxis with few side effects. PMID:26150717

  18. Key Role of CRF in the Skin Stress Response System

    PubMed Central

    Zmijewski, Michal A.; Zbytek, Blazej; Tobin, Desmond J.; Theoharides, Theoharis C.; Rivier, Jean

    2013-01-01

    The discovery of corticotropin-releasing factor (CRF) or CRH defining the upper regulatory arm of the hypothalamic-pituitary-adrenal (HPA) axis, along with the identification of the corresponding receptors (CRFRs 1 and 2), represents a milestone in our understanding of central mechanisms regulating body and local homeostasis. We focused on the CRF-led signaling systems in the skin and offer a model for regulation of peripheral homeostasis based on the interaction of CRF and the structurally related urocortins with corresponding receptors and the resulting direct or indirect phenotypic effects that include regulation of epidermal barrier function, skin immune, pigmentary, adnexal, and dermal functions necessary to maintain local and systemic homeostasis. The regulatory modes of action include the classical CRF-led cutaneous equivalent of the central HPA axis, the expression and function of CRF and related peptides, and the stimulation of pro-opiomelanocortin peptides or cytokines. The key regulatory role is assigned to the CRFR-1α receptor, with other isoforms having modulatory effects. CRF can be released from sensory nerves and immune cells in response to emotional and environmental stressors. The expression sequence of peptides includes urocortin/CRF→pro-opiomelanocortin→ACTH, MSH, and β-endorphin. Expression of these peptides and of CRFR-1α is environmentally regulated, and their dysfunction can lead to skin and systemic diseases. Environmentally stressed skin can activate both the central and local HPA axis through either sensory nerves or humoral factors to turn on homeostatic responses counteracting cutaneous and systemic environmental damage. CRF and CRFR-1 may constitute novel targets through the use of specific agonists or antagonists, especially for therapy of skin diseases that worsen with stress, such as atopic dermatitis and psoriasis. PMID:23939821

  19. Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

    NASA Astrophysics Data System (ADS)

    Seaton, D. D.; Krishnan, J.

    2012-08-01

    Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.

  20. Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

    PubMed

    Suwannarangsee, Surisa; Arnthong, Jantima; Eurwilaichitr, Lily; Champreda, Verawat

    2014-10-01

    Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.

  1. Robust enzyme design: bioinformatic tools for improved protein stability.

    PubMed

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA 1535/pSK1002.

    PubMed

    Shimada, T; Iwasaki, M; Martin, M V; Guengerich, F P

    1989-06-15

    A total of 57 procarcinogens was examined for induction of umu gene response in the chimeric plasmid pSK1002, carried in Salmonella typhimurium TA 1535, after incubation with a series of human liver microsomal preparations which had been selected on the basis of characteristic levels of individual cytochrome P-450 (P-450) enzymes. The 18 most active compounds were selected and further analyzed using the umu gene response and correlative studies with a larger number of microsomal preparations, enzyme reconstitution studies involving purified enzymes, immunochemical inhibition, and patterns of stimulation and inhibition of catalytic activity by 7,8-benzoflavone. The results collectively indicate that 16 of these 18 most potent genotoxins examined are activated primarily either by P-450NF (the nifedipine oxidase) or P-450PA (the phenacetin O-deethylase). P-450NF appears to be the major enzyme involved in the bioactivation of aflatoxin B1, aflatoxin G1, sterigmatocystin, trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, 6-aminochrysene, and tris-(2,3-dibromopropyl)phosphate in human liver. P-450PA appears to be the major enzyme involved in the bioactivation of 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl, 3-amino-1-methyl-5H-pyrido[4,3-b] indole, and 2-aminodipyrido[1,2-a:3',2'-d]imidazole. More than one enzyme appears to contribute significantly to the bioactivation of the other two compounds examined, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole and 6-nitrochrysene. The literature suggests that the two human liver P-450s involved in activation of these 16 procarcinogens are highly inducible by barbiturates, macrolide antibodies, and certain steroids (P-450NF) and by smoking and ingestion of charcoal-containing food (P-450PA); noninvasive assays are available

  3. hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis

    PubMed Central

    Park, Sun-Ji; Kim, Tae-Shin; Park, Choon-Keun; Lee, Sang-Hee; Kim, Jin-Man; Lee, Kyu-Sun; Lee, In-kyu; Park, Jeen-Woo; Lawson, Mark A; Lee, Dong-Seok

    2014-01-01

    Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. PMID:23256993

  4. Guest-Release Control in Enzyme-Sensitive, Amphiphilic-Dendrimer-Based Nanoparticles through Photochemical Crosslinking

    PubMed Central

    Raghupathi, Krishna R.; Azagarsamy, Malar A.; Thayumanavan, S.

    2012-01-01

    Stimuli sensitive, facially amphiphilic dendrimers have been synthesized and their enzyme-responsive nature has been determined with dual fluorescence responses of both covalently conjugated and non-covalently bound reporter units. These dual responses are correlated to ascertain the effect of enzymatic action on micellar aggregates and the consequential guest release. The release of the guest molecule is conveniently tuned by stabilizing the micellar aggregates through photochemical crosslinking of hydrophobic coumarin units. This photo-crosslinking is also utilized as a tool to investigate the mode of enzyme-substrate interaction in the context of aggregate-monomer equilibrium. PMID:21887830

  5. Assessment of maternal antibody decay and response to canine parvovirus vaccination using a clinic-based enzyme-linked immunosorbent assay.

    PubMed

    Waner, T; Naveh, A; Wudovsky, I; Carmichael, L E

    1996-10-01

    Interference caused by maternal antibodies is considered a major cause of canine parvovirus (CPV) vaccination failure. In this study, an immunoblot clinic-based enzyme-linked immunosorbent assay (ELISA) method was used to detect CPV antibodies in sera of pregnant bitches and their offspring to study the response of pups to vaccination. With a easily accessible procedure for CPV antibody determination, the veterinarian should be able to gauge the response of pups after vaccination. The validity of the technique was tested in parallel against the standard hemagglutination inhibition (HI) test. Results of the ELISA were correlated with those of the standard HI method for quantification of CPV antibodies. With the ELISA, successfully immunized pups were identified, allowing for a more reliable and cost-effective program of vaccination. This simple clinic-based test could be used for the assessment of vaccination status of pups during the critical phase of 6 to about 16 weeks of age. This study is the first in which vaccination response to CPV in pups was followed, using a clinic-based ELISA for CPV antibody monitoring.

  6. Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil

    PubMed Central

    Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

    2016-01-01

    Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility. PMID:27589265

  7. The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane.

    PubMed

    Scheller, Silvan; Goenrich, Meike; Boecher, Reinhard; Thauer, Rudolf K; Jaun, Bernhard

    2010-06-03

    Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.

  8. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

    PubMed

    Baston, Eckhard; Leroux, Frédéric R

    2007-01-01

    Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

  9. Born-Oppenheimer ab initio QM/MM Molecular Dynamics Simulations of Enzyme Reactions

    PubMed Central

    Zhou, Yanzi; Wang, Shenglong; Li, Yongle; Zhang, Yingkai

    2016-01-01

    There are two key requirements for reliably simulating enzyme reactions: one is a reasonably accurate potential energy surface to describe the bond forming/breaking process as well as to adequately model the heterogeneous enzyme environment; the other is to perform extensive sampling since an enzyme system consists of at least thousands of atoms and its energy landscape is very complex. One attractive approach to meet both daunting tasks is Born-Oppenheimer ab initio QM/MM molecular dynamics simulation (aiQM/MM-MD) with umbrella sampling. In this chapter, we describe our recently developed pseudobond Q-Chem–Amber interface, which employs a combined electrostatic-mechanical embedding scheme with periodic boundary condition and the particle mesh Ewald method for long-range electrostatics interactions. In our implementation, Q-Chem and the sander module of Amber are combined at the source code level without using system calls, and all necessary data communications between QM and MM calculations are achieved via computer memory. We demonstrate the applicability of this pseudobond Q-Chem–Amber interface by presenting two examples, one reaction in aqueous solution and one enzyme reaction. Finally, we describe our established aiQM/MM-MD enzyme simulation protocol, which has been successfully applied to study more than a dozen enzymes. PMID:27498636

  10. NNT is a key regulator of adrenal redox homeostasis and steroidogenesis in male mice

    PubMed Central

    Goldsworthy, M; Chortis, V; Fragouli, E; Foster, P A; Arlt, W; Cox, R; Metherell, L A

    2018-01-01

    Nicotinamide nucleotide transhydrogenase, NNT, is a ubiquitous protein of the inner mitochondrial membrane with a key role in mitochondrial redox balance. NNT produces high concentrations of NADPH for detoxification of reactive oxygen species by glutathione and thioredoxin pathways. In humans, NNT dysfunction leads to an adrenal-specific disorder, glucocorticoid deficiency. Certain substrains of C57BL/6 mice contain a spontaneously occurring inactivating Nnt mutation and display glucocorticoid deficiency along with glucose intolerance and reduced insulin secretion. To understand the underlying mechanism(s) behind the glucocorticoid deficiency, we performed comprehensive RNA-seq on adrenals from wild-type (C57BL/6N), mutant (C57BL/6J) and BAC transgenic mice overexpressing Nnt (C57BL/6JBAC). The following results were obtained. Our data suggest that Nnt deletion (or overexpression) reduces adrenal steroidogenic output by decreasing the expression of crucial, mitochondrial antioxidant (Prdx3 and Txnrd2) and steroidogenic (Cyp11a1) enzymes. Pathway analysis also revealed upregulation of heat shock protein machinery and haemoglobins possibly in response to the oxidative stress initiated by NNT ablation. In conclusion, using transcriptomic profiling in adrenals from three mouse models, we showed that disturbances in adrenal redox homeostasis are mediated not only by under expression of NNT but also by its overexpression. Further, we demonstrated that both under expression or overexpression of NNT reduced corticosterone output implying a central role for it in the control of steroidogenesis. This is likely due to a reduction in the expression of a key steroidogenic enzyme, Cyp11a1, which mirrored the reduction in corticosterone output. PMID:29046340

  11. Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

    PubMed Central

    Nguyen, Minh Thu

    2016-01-01

    SUMMARY Since the discovery in 1973 of the first of the bacterial lipoproteins (Lpp) in Escherichia coli, Braun's lipoprotein, the ever-increasing number of publications indicates the importance of these proteins. Bacterial Lpp belong to the class of lipid-anchored proteins that in Gram-negative bacteria are anchored in both the cytoplasmic and outer membranes and in Gram-positive bacteria are anchored only in the cytoplasmic membrane. In contrast to the case for Gram-negative bacteria, in Gram-positive bacteria lipoprotein maturation and processing are not vital. Physiologically, Lpp play an important role in nutrient and ion acquisition, allowing particularly pathogenic species to better survive in the host. Bacterial Lpp are recognized by Toll-like receptor 2 (TLR2) of the innate immune system. The important role of Lpp in Gram-positive bacteria, particularly in the phylum Firmicutes, as key players in the immune response and pathogenicity has emerged only in recent years. In this review, we address the role of Lpp in signaling and modulating the immune response, in inflammation, and in pathogenicity. We also address the potential of Lpp as promising vaccine candidates. PMID:27512100

  12. Highlighting the Need for Systems-Level Experimental Characterization of Plant Metabolic Enzymes.

    PubMed

    Engqvist, Martin K M

    2016-01-01

    The biology of living organisms is determined by the action and interaction of a large number of individual gene products, each with specific functions. Discovering and annotating the function of gene products is key to our understanding of these organisms. Controlled experiments and bioinformatic predictions both contribute to functional gene annotation. For most species it is difficult to gain an overview of what portion of gene annotations are based on experiments and what portion represent predictions. Here, I survey the current state of experimental knowledge of enzymes and metabolism in Arabidopsis thaliana as well as eleven economically important crops and forestry trees - with a particular focus on reactions involving organic acids in central metabolism. I illustrate the limited availability of experimental data for functional annotation of enzymes in most of these species. Many enzymes involved in metabolism of citrate, malate, fumarate, lactate, and glycolate in crops and forestry trees have not been characterized. Furthermore, enzymes involved in key biosynthetic pathways which shape important traits in crops and forestry trees have not been characterized. I argue for the development of novel high-throughput platforms with which limited functional characterization of gene products can be performed quickly and relatively cheaply. I refer to this approach as systems-level experimental characterization. The data collected from such platforms would form a layer intermediate between bioinformatic gene function predictions and in-depth experimental studies of these functions. Such a data layer would greatly aid in the pursuit of understanding a multiplicity of biological processes in living organisms.

  13. Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

    DOEpatents

    Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

    1992-01-01

    An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

  14. Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

    DOEpatents

    Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

    1993-01-01

    An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

  15. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    PubMed

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  16. Efficacy ranking of triterpenoids as inducers of a cytoprotective enzyme and as inhibitors of a cellular inflammatory response via their electron affinity and their electrophilicity index

    PubMed Central

    Bensasson, René V.; Zoete, Vincent; Berthier, Gaston; Talalay, Paul; Dinkova-Kostova, Albena T.

    2010-01-01

    Electron affinity (EA) and electrophilicity index (ω) of 16 synthetic triterpenoids (TP), previously identified as inducers of cytoprotective enzymes and as inhibitors of cellular inflammatory responses, have been calculated by the molecular orbital method. Linear correlations were obtained by plotting the values of EA, as well as those of ω versus (i) the potencies of induction of NAD(P)H quinone reductase (NQO1, EC 1.6.99.2), a cytoprotective enzyme, expressed via the concentration of TP required to double the specific activity of NQO1 (CD value) and (ii) the values of their anti-inflammatory activity expressed via the IC-50 of TP for suppression of upregulation of inducible nitric oxide synthase (iNOS, EC 1.14.13.39), both previously experimentally determined. The observed correlations demonstrate quantitatively for a series of triterpenoids that their electrophilicity is a major factor determining their potency as inducers of the cytoprotective phase 2 response and as inhibitors of inflammatory processes. PMID:20433811

  17. Cloning of β-Primeverosidase from Tea Leaves, a Key Enzyme in Tea Aroma Formation1

    PubMed Central

    Mizutani, Masaharu; Nakanishi, Hidemitsu; Ema, Jun-ichi; Ma, Seung-Jin; Noguchi, Etsuko; Inohara-Ochiai, Misa; Fukuchi-Mizutani, Masako; Nakao, Masahiro; Sakata, Kanzo

    2002-01-01

    A β-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of β-primeverosides (6-O-β-d-xylopyranosyl-β-d-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The β-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The β-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The β-primeverosidase protein sequence was 50% to 60% identical to β-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the β-primeverosidase expressed in Escherichia coli was able to hydrolyze β-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl β-d-glucopyranoside. These results indicate that the β-primeverosidase selectively recognizes the β-primeverosides as substrates and specifically hydrolyzes the β-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl β-primeveroside by the β-primeverosidase was followed by 1H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the β-primeveroside by a retaining mechanism. The roles of the β-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed. PMID:12481100

  18. HPLC-DAD fingerprinting analysis, antioxidant activities of Tithonia diversifolia (Hemsl.) A. Gray leaves and its inhibition of key enzymes linked to Alzheimer's disease.

    PubMed

    Ojo, Oluwafemi Adeleke; Ojo, Adebola Busola; Ajiboye, Basiru Olaitan; Olaiya, Oluranti; Okesola, Mary Abiola; Boligon, Aline Augusti; de Campos, Marli Matiko Anraku; Oyinloye, Babatunji Emmanuel; Kappo, Abidemi Paul

    2018-01-01

    Tithonia diversifolia (Hemsl.) A. Gray leaves have long been used to manage neurodegenerative diseases without scientific basis. This study characterized the phenolic constituents, evaluated the antioxidant properties of phenolic extracts from T. diversifolia leaves used as traditional medicine in Africa and its inhibition of key enzymes linked to Alzheimer's disease. The extract was rich in phenolic acids (gallic acid, chlorogenic acid, caffeic acid and p -coumaric acid) and flavonoids (apigenin) and had 1,1-diphenyl-2-picryl-hydrazil radical scavenging abilities (IC 50  = 41.05 μg. mL -1 ), 2,2-Azino-bis3-ethylbenthiazoline-6sulphonic acid radical scavenging ability (IC 50  = 33.51 μg. mL -1 ), iron chelation (IC 50  = 38.50 μg. mL -1 ), reducing power (Fe 3+ - Fe 2+ ) (7.34 AAEmg/100 g), inhibited acetylcholinesterase (IC 50  = 39.27 μg mL -1 ) and butyrylcholinesterase (IC 50  = 35.01 μg mL -1 ) activities. These results reveal the leaf as a rich source of phenolic compounds with antioxidant and cholinesterase inhibitory activity.

  19. Phenolic-rich extracts of Eurycoma longifolia and Cylicodiscus gabunensis inhibit enzymes responsible for the development of erectile dysfunction and are antioxidants.

    PubMed

    Oboh, Ganiyu; Adebayo, Adeniyi A; Ademosun, Ayokunle O

    2018-05-19

    Herbs have been used from ages to manage male sexual dysfunction. Hence, this study sought to investigate the effects of Eurycoma longifolia (EL) and Cylicodiscus gabunensis (CG) stem bark extracts on some enzymes implicated in erectile dysfunction in vitro. The extracts were prepared, and their effects on phosphodiesterase-5 (PDE-5), arginase, and angiotensin-1-converting enzyme (ACE) as well as pro-oxidant-induced lipid peroxidation were assessed. Furthermore, phenolic contents were determined, and their components were characterized and quantified using high-performance liquid chromatography with diode array detector (HPLC-DAD). The results revealed that the extracts inhibited PDE-5, arginase, and ACE in a concentration-dependent manner. However, IC50 values revealed that CG had higher inhibitory potential on PDE-5 (IC50=204.4 μg/mL), arginase (IC50=39.01 μg/mL), and ACE (IC50=48.81 μg/mL) than EL. In addition, the extracts inhibited pro-oxidant-induced lipid peroxidation in penile tissue homogenate. HPLC-DAD analysis showed that CG is richer in phenolic compounds than EL, and this could be responsible for higher biological activities observed in CG than EL. Hence, the observed antioxidant property and inhibitory action of CG and EL on enzymes relevant to erectile dysfunction in vitro could be part of possible mechanisms underlying their involvement in traditional medicine for the management of male sexual dysfunction.

  20. OsGA2ox5, a Gibberellin Metabolism Enzyme, Is Involved in Plant Growth, the Root Gravity Response and Salt Stress

    NASA Astrophysics Data System (ADS)

    Cai, Weiming; Shan, Chi

    Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2b-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GAdeficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 mM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.

  1. OsGA2ox5, a Gibberellin Metabolism Enzyme, Is Involved in Plant Growth, the Root Gravity Response and Salt Stress

    PubMed Central

    Shan, Chi; Mei, Zhiling; Duan, Jianli; Chen, Haiying; Feng, Huafeng; Cai, Weiming

    2014-01-01

    Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice. PMID:24475234

  2. OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress.

    PubMed

    Shan, Chi; Mei, Zhiling; Duan, Jianli; Chen, Haiying; Feng, Huafeng; Cai, Weiming

    2014-01-01

    Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C₁₉-GA2oxs and a smaller class of C₂₀-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C₂₀-GA2oxs subfamily, a subfamily of GA2oxs acting on C₂₀-GAs (GA₁₂, GA₅₃). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.

  3. Enzyme Histochemistry for Functional Histology in Invertebrates.

    PubMed

    Cima, Francesca

    2017-01-01

    In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.

  4. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tolar, Bradley B.; Herrmann, Jonathan; Bargar, John R.

    In this paper, knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowlede of key N-cycling enzymes from ecologically-relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, though (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) andmore » nitrite reductase (NirK).« less

  5. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea

    DOE PAGES

    Tolar, Bradley B.; Herrmann, Jonathan; Bargar, John R.; ...

    2017-07-05

    In this paper, knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowlede of key N-cycling enzymes from ecologically-relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, though (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) andmore » nitrite reductase (NirK).« less

  6. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea.

    PubMed

    Tolar, Bradley B; Herrmann, Jonathan; Bargar, John R; van den Bedem, Henry; Wakatsuki, Soichi; Francis, Christopher A

    2017-10-01

    Knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowledge of key N-cycling enzymes from ecologically relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, through (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) and nitrite reductase (NirK). © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    NASA Astrophysics Data System (ADS)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  8. Scaffoldless engineered enzyme assembly for enhanced methanol utilization

    DOE PAGES

    Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

    2016-10-24

    Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

  9. Scaffoldless engineered enzyme assembly for enhanced methanol utilization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Price, J. Vincent; Chen, Long; Whitaker, W. Brian

    Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

  10. Scaffoldless engineered enzyme assembly for enhanced methanol utilization

    PubMed Central

    Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

    2016-01-01

    Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. PMID:27791059

  11. RSA-Based Password-Authenticated Key Exchange, Revisited

    NASA Astrophysics Data System (ADS)

    Shin, Seonghan; Kobara, Kazukuni; Imai, Hideki

    The RSA-based Password-Authenticated Key Exchange (PAKE) protocols have been proposed to realize both mutual authentication and generation of secure session keys where a client is sharing his/her password only with a server and the latter should generate its RSA public/private key pair (e, n), (d, n) every time due to the lack of PKI (Public-Key Infrastructures). One of the ways to avoid a special kind of off-line (so called e-residue) attacks in the RSA-based PAKE protocols is to deploy a challenge/response method by which a client verifies the relative primality of e and φ(n) interactively with a server. However, this kind of RSA-based PAKE protocols did not give any proof of the underlying challenge/response method and therefore could not specify the exact complexity of their protocols since there exists another security parameter, needed in the challenge/response method. In this paper, we first present an RSA-based PAKE (RSA-PAKE) protocol that can deploy two different challenge/response methods (denoted by Challenge/Response Method1 and Challenge/Response Method2). The main contributions of this work include: (1) Based on the number theory, we prove that the Challenge/Response Method1 and the Challenge/Response Method2 are secure against e-residue attacks for any odd prime e (2) With the security parameter for the on-line attacks, we show that the RSA-PAKE protocol is provably secure in the random oracle model where all of the off-line attacks are not more efficient than on-line dictionary attacks; and (3) By considering the Hamming weight of e and its complexity in the. RSA-PAKE protocol, we search for primes to be recommended for a practical use. We also compare the RSA-PAKE protocol with the previous ones mainly in terms of computation and communication complexities.

  12. A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals.

    PubMed

    Herrera, Bobby Brooke; Hamel, Donald J; Oshun, Philip; Akinsola, Rolake; Akanmu, Alani S; Chang, Charlotte A; Eromon, Philomena; Folarin, Onikepe; Adeyemi, Kayode T; Happi, Christian T; Lu, Yichen; Ogunsola, Folasade; Kanki, Phyllis J

    2018-05-01

    Ebola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

  13. Sodium and Potassium Ions in Proteins and Enzyme Catalysis.

    PubMed

    Vašák, Milan; Schnabl, Joachim

    2016-01-01

    The group I alkali metal ions Na(+) and K(+) are ubiquitous components of biological fluids that surround biological macromolecules. They play important roles other than being nonspecific ionic buffering agents or mediators of solute exchange and transport. Molecular evolution and regulated high intracellular and extracellular M(+) concentrations led to incorporation of selective Na(+) and K(+) binding sites into enzymes to stabilize catalytic intermediates or to provide optimal positioning of substrates. The mechanism of M(+) activation, as derived from kinetic studies along with structural analysis, has led to the classification of cofactor-like (type I) or allosteric effector (type II) activated enzymes. In the type I mechanism substrate anchoring to the enzyme active site is mediated by M(+), often acting in tandem with a divalent cation like Mg(2+), Mn(2+) or Zn(2+). In the allosteric type II mechanism, M(+) binding enhances enzyme activity through conformational transitions triggered upon binding to a distant site. In this chapter, following the discussion of the coordination chemistry of Na(+) and K(+) ions and the structural features responsible for the metal binding site selectivity in M(+)-activated enzymes, well-defined examples of M(+)-activated enzymes are used to illustrate the structural basis for type I and type II activation by Na(+) and K(+).

  14. Drought response in wheat: key genes and regulatory mechanisms controlling root system architecture and transpiration efficiency

    NASA Astrophysics Data System (ADS)

    Kulkarni, Manoj; Soolanayakanahally, Raju; Ogawa, Satoshi; Uga, Yusaku; Selvaraj, Michael G.; Kagale, Sateesh

    2017-12-01

    Abiotic stresses such as drought, heat, salinity and flooding threaten global food security. Crop genetic improvement with increased resilience to abiotic stresses is a critical component of crop breeding strategies. Wheat is an important cereal crop and a staple food source globally. Enhanced drought tolerance in wheat is critical for sustainable food production and global food security. Recent advances in drought tolerance research have uncovered many key genes and transcription regulators governing morpho-physiological traits. Genes controlling root architecture and stomatal development play an important role in soil moisture extraction and its retention, and therefore have been targets of molecular breeding strategies for improving drought tolerance. In this systematic review, we have summarized evidence of beneficial contributions of root and stomatal traits to plant adaptation to drought stress. Specifically, we discuss a few key genes such as DRO1 in rice and ERECTA in Arabidopsis and rice that were identified to be the enhancers of drought tolerance via regulation of root traits and transpiration efficiency. Additionally, we highlight several transcription factor families, such as ERF (ethylene response factors), DREB (dehydration responsive element binding), ZFP (zinc finger proteins), WRKY and MYB that were identified to be both positive and negative regulators of drought responses in wheat, rice, maize and/or Arabidopsis. The overall aim of this review was to provide an overview of candidate genes that have been tested as regulators of drought response in plants. The lack of a reference genome sequence for wheat and nontransgenic approaches for manipulation of gene functions in the past had impeded high-resolution interrogation of functional elements, including genes and QTLs, and their application in cultivar improvement. The recent developments in wheat genomics and reverse genetics, including the availability of a gold-standard reference genome

  15. Drought Response in Wheat: Key Genes and Regulatory Mechanisms Controlling Root System Architecture and Transpiration Efficiency.

    PubMed

    Kulkarni, Manoj; Soolanayakanahally, Raju; Ogawa, Satoshi; Uga, Yusaku; Selvaraj, Michael G; Kagale, Sateesh

    2017-01-01

    Abiotic stresses such as, drought, heat, salinity, and flooding threaten global food security. Crop genetic improvement with increased resilience to abiotic stresses is a critical component of crop breeding strategies. Wheat is an important cereal crop and a staple food source globally. Enhanced drought tolerance in wheat is critical for sustainable food production and global food security. Recent advances in drought tolerance research have uncovered many key genes and transcription regulators governing morpho-physiological traits. Genes controlling root architecture and stomatal development play an important role in soil moisture extraction and its retention, and therefore have been targets of molecular breeding strategies for improving drought tolerance. In this systematic review, we have summarized evidence of beneficial contributions of root and stomatal traits to plant adaptation to drought stress. Specifically, we discuss a few key genes such as, DRO1 in rice and ERECTA in Arabidopsis and rice that were identified to be the enhancers of drought tolerance via regulation of root traits and transpiration efficiency. Additionally, we highlight several transcription factor families, such as, ERF (ethylene response factors), DREB (dehydration responsive element binding), ZFP (zinc finger proteins), WRKY, and MYB that were identified to be both positive and negative regulators of drought responses in wheat, rice, maize, and/or Arabidopsis. The overall aim of this review is to provide an overview of candidate genes that have been identified as regulators of drought response in plants. The lack of a reference genome sequence for wheat and non-transgenic approaches for manipulation of gene functions in wheat in the past had impeded high-resolution interrogation of functional elements, including genes and QTLs, and their application in cultivar improvement. The recent developments in wheat genomics and reverse genetics, including the availability of a gold

  16. Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

    PubMed

    Ahmed, Nisar; Tetlow, Ian J; Nawaz, Sehar; Iqbal, Ahsan; Mubin, Muhammad; Nawaz ul Rehman, Muhammad Shah; Butt, Aisha; Lightfoot, David A; Maekawa, Masahiko

    2015-08-30

    High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis. © 2014 Society of Chemical Industry.

  17. Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair

    PubMed Central

    Bhat, Javaid Y.; Thieulin-Pardo, Gabriel; Hartl, F. Ulrich; Hayer-Hartl, Manajit

    2017-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO2 molecule and binding of a Mg2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are “misfire” products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins. PMID:28443288

  18. Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair.

    PubMed

    Bhat, Javaid Y; Thieulin-Pardo, Gabriel; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2017-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO 2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO 2 molecule and binding of a Mg 2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

  19. Of enzyme use in cost-effective high solid simultaneous saccharification and fermentation processes.

    PubMed

    Sóti, Valentin; Lenaerts, Silvia; Cornet, Iris

    2018-03-20

    Enzyme cost is considered to be one of the most significant factors defining the final product price in lignocellulose hydrolysis and fermentation. Enzyme immobilization and recycling can be a tool to decrease costs. However, high solid loading is a key factor towards high product titers, and recovery of immobilized enzymes from this thick liquid is often overlooked. This paper aims to evaluate the economic feasibility of immobilized enzymes in simultaneous saccharification and fermentation (SSF) of lignocellulose biomass in general, as well as the recuperation of magnetic immobilized enzymes (m-CLEAs) during high solid loading in simultaneous saccharification, detoxification and fermentation processes (SSDF) of lignocellulose biomass. Enzyme prices were obtained from general cost estimations by Klein-Marcuschamer et al. [Klein-Marcuschamer et al. (2012) Biotechnol. Bioeng. 109, 1083-1087]. During enzyme cost analysis, the influence of inoculum recirculation as well as a shortened fermentation time was explored. Both resulted in 15% decrease of final enzyme product price. Enzyme recuperation was investigated experimentally and 99.5 m/m% of m-CLEAs was recovered from liquid medium in one step, while 88 m/m% could still be recycled from a thick liquid with high solid concentrations (SSF fermentation broth). A mathematical model was constructed to calculate the cost of immobilized and free enzyme utilization and showed that, with current process efficiencies and commercial enzyme prices, the cost reduction obtained by enzyme immobilization can reach around 60% compared to free enzyme utilization, while lower enzyme prices will result in a lower percentage of immobilization related savings, but overall enzyme costs will decrease significantly. These results are applied in a case study, estimating the viability of shifting from sugar to lignocellulose substrate for a 100 t lactic acid fermentation batch. It was concluded that it will only be economically feasible

  20. Temperature Sensitivities of Extracellular Enzyme Vmax and Km Across Thermal Environments

    NASA Astrophysics Data System (ADS)

    Allison, S. D.; Romero-Olivares, A.; Lu, Y.; Taylor, J.; Treseder, K. K.

    2017-12-01

    The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivity of microbial processes in soil. Enzymatic rates could increase at higher temperatures, but this response is determined by multiple parameters that may change over time if soil microbes adapt to warming. We used the Michaelis-Menten relationship, the Arrhenius relationship, and biochemical transition state theory to construct hypotheses about the responses of extracellular enzyme Vmax and Km to temperature. Based on the Arrhenius relationship, we hypothesized that Vmax and Km would show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower Vmax, Km, and Km temperature sensitivity but higher Vmax temperature sensitivity. We tested these hypotheses with enzymes from isolates of the filamentous fungus Neurospora discreta collected around the globe and from decomposing leaf litter in a warming experiment in Alaskan boreal forest. Vmax and Km of most Neurospora extracellular enzymes were temperature sensitive with average Vmax Q10 ranging from 1.48 to 2.25 and Km Q10 ranging from 0.71 to 2.80. For both Vmax and Km, there was a tendency for the parameter to correlate negatively with its temperature sensitivity, a pattern predicted by biochemical theory. Also in agreement with theory, Vmax and Km were positively correlated for some enzymes. In contrast, there was little support for biochemical theory when comparing Vmax and Km across thermal environments. There was no relationship between temperature sensitivity of Vmax or Km and mean annual temperature of the isolation site for Neurospora strains. There was some evidence for greater Vmax under experimental warming in Alaskan litter, but the temperature sensitivities of Vmax and Km did not vary with warming as expected. We conclude that relationships among Vmax, Km, and temperature are largely consistent with biochemical

  1. Fluorescent carbon dot-gated multifunctional mesoporous silica nanocarriers for redox/enzyme dual-responsive targeted and controlled drug delivery and real-time bioimaging.

    PubMed

    Wang, Ying; Cui, Yu; Zhao, Yating; He, Bing; Shi, Xiaoli; Di, Donghua; Zhang, Qiang; Wang, Siling

    2017-08-01

    A distinctive and personalized nanocarrier is described here for controlled and targeted antitumor drug delivery and real-time bioimaging by combining a redox/enzyme dual-responsive disulfide-conjugated carbon dot with mesoporous silica nanoparticles (MSN-SS-CD HA ). The carbon dot with controlling and targeting abilities was prepared through a polymerizing reaction by applying citric acid and HA as starting materials (named CD HA ). The as-prepared MSN-SS-CD HA exhibited not only superior photostability and excellent biocompatibility, but also the ability to target A549 cells with overexpression of CD44 receptors. Upon loading the antitumor drug, doxorubicin (DOX), into the mesoporous channels of MSN nanoparticles, CD HA with a diameter size of 3nm completely blocked the pore entrance of DOX-encapsulated MSN nanoparticles with a pore size of about 3nm, thus preventing the premature leakage of DOX and increasing the antitumor activity until being triggered by specific stimuli in the tumor environment. The results of the cell imaging and cytotoxicity studies demonstrated that the redox/enzyme dual-responsive DOX-encapsulated MSN-SS-CD HA nanoparticles can selectively deliver and control the release of DOX into tumor cells. Ex vivo fluorescence images showed a much stronger fluorescence of MSN-SS-CD HA -DOX in the tumor site than in normal tissues, greatly facilitating the accumulation of DOX in the target tissue. However, its counterpart, MSN-SH-DOX exhibited no or much lower tumor cytotoxicity and drug accumulation in tumor tissue. In addition, MSN-SS-CD was also used as a control to investigate the ability of MSN-SS-CD HA to target A549 cells. The results obtained indicated that MSN-SS-CD HA possessed a higher cellular uptake through the CD44 receptor-mediated endocytosis compared with MSN-SS-CD in the A549 cells. Such specific redox/enzyme dual-responsive targeted nanocarriers are a useful strategy achieving selective controlled and targeted delivery of

  2. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    PubMed

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

  3. Vacuolar processing enzyme in plant programmed cell death

    PubMed Central

    Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

    2015-01-01

    Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

  4. Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes.

    PubMed

    Jacquet, Pauline; Daudé, David; Bzdrenga, Janek; Masson, Patrick; Elias, Mikael; Chabrière, Eric

    2016-05-01

    Organophosphorus chemicals are highly toxic molecules mainly used as pesticides. Some of them are banned warfare nerve agents. These compounds are covalent inhibitors of acetylcholinesterase, a key enzyme in central and peripheral nervous systems. Numerous approaches, including chemical, physical, and biological decontamination, have been considered for developing decontamination methods against organophosphates (OPs). This work is an overview of both validated and emerging strategies for the protection against OP pollution with special attention to the use of decontaminating enzymes. Considerable efforts have been dedicated during the past decades to the development of efficient OP degrading biocatalysts. Among these, the promising biocatalyst SsoPox isolated from the archaeon Sulfolobus solfataricus is emphasized in the light of recently published results. This hyperthermostable enzyme appears to be particularly attractive for external decontamination purposes with regard to both its catalytic and stability properties.

  5. Scaffolding Function of PI3Kgamma Emerges from Enzyme's Shadow.

    PubMed

    Mohan, Maradumane L; Naga Prasad, Sathyamangla V

    2017-03-24

    Traditionally, an enzyme is a protein that mediates biochemical action by binding to the substrate and by catalyzing the reaction that translates external cues into biological responses. Sequential dissemination of information from one enzyme to another facilitates signal transduction in biological systems providing for feed-forward and feed-back mechanisms. Given this viewpoint, an enzyme without its catalytic activity is generally considered to be an inert organizational protein without catalytic function and has classically been termed as pseudo-enzymes. However, pseudo-enzymes still have biological function albeit non-enzymatic like serving as a chaperone protein or an interactive platform between proteins. In this regard, majority of the studies have focused solely on the catalytic role of enzymes in biological function, overlooking the potentially critical non-enzymatic roles. Increasing evidence from recent studies implicate that the scaffolding function of enzymes could be as important in signal transduction as its catalytic activity, which is an antithesis to the definition of enzymes. Recognition of non-enzymatic functions could be critical, as these unappreciated roles may hold clues to the ineffectiveness of kinase inhibitors in pathology, which is characteristically associated with increased enzyme expression. Using an established enzyme phosphoinositide 3-kinase γ, we discuss the insights obtained from the scaffolding function and how this non-canonical role could contribute to/alter the outcomes in pathology like cancer and heart failure. Also, we hope that with this review, we provide a forum and a starting point to discuss the idea that catalytic function alone may not account for all the actions observed with increased expression of the enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

    PubMed

    Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

    2017-08-03

    Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

  7. Gonadal development and transcript profiling of steroidogenic enzymes in response to 17α-methyltestosterone in the rare minnow Gobiocypris rarus.

    PubMed

    Liu, Shaozhen; Wang, Lihong; Qin, Fang; Zheng, Yao; Li, Meng; Zhang, Yingying; Yuan, Cong; Wang, Zaizhao

    2014-09-01

    It is well known that natural and anthropogenic chemicals interfere with the hormonal system of vertebrate and invertebrate organisms. How these chemicals regulate gonadal steroidogenesis remains to be determined. The main objective of this study was to evaluate the effects of 17α-methyltestosterone (MT), a synthetic model androgen, on gene expression profiles of six key steroidogenic genes in adult rare minnow. The full-length cDNA encoding 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) was firstly isolated and characterized by RT-PCR and RACE methods. The gonadal transcript changes of StAR, cyp11a1, 3β-HSD, cyp17a1, 11β-HSD2 and cyp19a1a in 6-month adult Gobiocypris rarus exposed to MT and 17α-ethinylestradiol (EE2) for 7, 14 and 21 days were detected by qRT-PCR. To make an effort to connect the transcriptional changes of steroidogenic enzymes with effects on higher levels of biological organization and on VTG, one remarkable sensitive target of steroids, body and gonad weights, histology of gonads, and hepatic vtg mRNA level were measured. MT caused varying degree of abnormalities in ovaries and testes. The hepatic vtg mRNA level was highly inhibited in females and slightly altered in males by MT. Transcripts of several steroidogenic genes including StAR, cyp17a1, and cyp11a1 showed high responsiveness to MT exposure in G. rarus. The gene expression profiles of these steroidogenic genes in MT-treated groups were much distinct with the EE2-treated group. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Enzyme replacement and substrate reduction therapy for Gaucher disease.

    PubMed

    Shemesh, Elad; Deroma, Laura; Bembi, Bruno; Deegan, Patrick; Hollak, Carla; Weinreb, Neal J; Cox, Timothy M

    2015-03-27

    Gaucher disease, a rare disorder, is caused by inherited deficiency of the enzyme glucocerebrosidase. It is unique among the ultra-orphan disorders in that four treatments are currently approved by various regulatory authorities for use in routine clinical practice. Hitherto, because of the relatively few people affected worldwide, many of whom started therapy during a prolonged period when there were essentially no alternatives to imiglucerase, these treatments have not been systematically evaluated in studies such as randomized controlled trials now considered necessary to generate the highest level of clinical evidence. To summarize all available randomized controlled study data on the efficacy and safety of enzyme replacement therapies and substrate reduction therapy for treating Gaucher disease. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Inborn Errors of Metabolism Trials Register. Additional searches were conducted on ClinicalTrials.gov for any ongoing studies with potential interim results, and through PubMed. We also searched the reference lists of relevant articles and reviews.Date of last search: 07 August 2014. All randomized and quasi-randomized controlled studies (including open-label studies and cross-over studies) assessing enzyme replacement therapy or substrate reduction therapy, or both, in all types of Gaucher disease were included. Two authors independently assessed the risk of bias in the included studies, and extracted relevant data. Of the 488 studies retrieved by the electronic searches, eight met the inclusion criteria and were analysed (300 participants). Response parameters were restricted to haemoglobin concentration, platelet count, spleen and liver volume and serum biomarkers (chitotriosidase and CCL18). Only one publication reported a 'low risk of bias' score in all parameters assessed, and all studies included were randomized.Four studies reported the responses to enzyme replacement therapy of previously

  9. Human Chitotriosidase Is an Endo-Processive Enzyme

    PubMed Central

    Sørlie, Morten; Väljamäe, Priit

    2017-01-01

    Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found–the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT. PMID:28129403

  10. Genetic variation in the urea cycle: a model resource for investigating key candidate genes for common diseases.

    PubMed

    Mitchell, Sabrina; Ellingson, Clint; Coyne, Thomas; Hall, Lynn; Neill, Meaghan; Christian, Natalie; Higham, Catherine; Dobrowolski, Steven F; Tuchman, Mendel; Summar, Marshall

    2009-01-01

    The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases. Copyright 2008 Wiley-Liss, Inc.

  11. Substance P increases Sympathetic Activity during Combined Angiotensin Converting Enzyme and Dipeptidyl Peptidase-4 Inhibition

    PubMed Central

    Devin, Jessica K.; Pretorius, Mias; Nian, Hui; Yu, Chang; Billings, Frederic T.; Brown, Nancy J.

    2014-01-01

    Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce post-prandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme. During angiotensin-converting enzyme inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, while bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when angiotensin-converting enzyme is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg p.o. or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat, however, substance P increased heart rate and vascular release of norepinephrine during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition. PMID:24516103

  12. DNA based random key generation and management for OTP encryption.

    PubMed

    Zhang, Yunpeng; Liu, Xin; Sun, Manhui

    2017-09-01

    One-time pad (OTP) is a principle of key generation applied to the stream ciphering method which offers total privacy. The OTP encryption scheme has proved to be unbreakable in theory, but difficult to realize in practical applications. Because OTP encryption specially requires the absolute randomness of the key, its development has suffered from dense constraints. DNA cryptography is a new and promising technology in the field of information security. DNA chromosomes storing capabilities can be used as one-time pad structures with pseudo-random number generation and indexing in order to encrypt the plaintext messages. In this paper, we present a feasible solution to the OTP symmetric key generation and transmission problem with DNA at the molecular level. Through recombinant DNA technology, by using only sender-receiver known restriction enzymes to combine the secure key represented by DNA sequence and the T vector, we generate the DNA bio-hiding secure key and then place the recombinant plasmid in implanted bacteria for secure key transmission. The designed bio experiments and simulation results show that the security of the transmission of the key is further improved and the environmental requirements of key transmission are reduced. Analysis has demonstrated that the proposed DNA-based random key generation and management solutions are marked by high security and usability. Published by Elsevier B.V.

  13. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    PubMed

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  14. Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

    NASA Astrophysics Data System (ADS)

    Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

    It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

  15. Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.

    PubMed

    Styles, Matthew Q; Nesbitt, Edward A; Marr, Scott; Hutchby, Marc; Leak, David J

    2017-06-01

    The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class-I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ-muurolol at temperatures up to 78 °C. We also report an enzyme with a k cat value of 0.95 s -1 at 65 °C, the highest k cat recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class-I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. Model data are available in the PMDB database under the accession number PM0080780. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  16. NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis.

    PubMed

    Raad, Houssam; Serrano-Sanchez, Martin; Harfouche, Ghida; Mahfouf, Walid; Bortolotto, Doriane; Bergeron, Vanessa; Kasraian, Zeinab; Dousset, Lea; Hosseini, Mohsen; Taieb, Alain; Rezvani, Hamid Reza

    2017-06-01

    The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  18. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  19. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  20. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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