Letterman Army Institute of Research Annual Research Progress Report, FY 1981.

DTIC Science & Technology

1981-10-01

with physiology and mech- anisms of skin damage and repair. The mechanisms by which nerve agents and vesicants produce physiologic aberration and...enzyme vital for nerve function. Organophos- phates react rapidly and covalently with the enzyme to produce an in- active enzyme. Reactivation of the...warfare agents . These organic molecules may also alter the natural defense mechanism by activating or deactivating enzymes in the skin that destroy

Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

PubMed

Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

2014-10-01

Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

PubMed

Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

2014-12-01

Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Effect of Sodium Fluoride Ingestion on Malondialdehyde Concentration and the Activity of Antioxidant Enzymes in Rat Erythrocytes

PubMed Central

Morales-González, José A.; Gutiérrez-Salinas, José; García-Ortiz, Liliana; del Carmen Chima-Galán, María; Madrigal-Santillán, Eduardo; Esquivel-Soto, Jaime; Esquivel-Chirino, César; González-Rubio, Manuel García-Luna y

2010-01-01

Fluoride intoxication has been shown to produce diverse deleterious metabolic alterations within the cell. To determine the effects of sodium fluoride (NaF) treatment on malondialdehyde (MDA) levels and on the activity of antioxidant enzymes in rat erythrocytes, Male Wistar rats were treated with 50 ppm of NaF or were untreated as controls. Erythrocytes were obtained from rats sacrificed weekly for up to eight weeks and the concentration of MDA in erythrocyte membrane was determined. In addition, the activity of the enzymes superoxide, dismutase, catalase, and glutathione peroxidase were determined. Treatment with NaF produces an increase in the concentration of malondialdehyde in the erythrocyte membrane only after the eight weeks of treatment. On the other hand, antioxidant enzyme activity was observed to increase after the fourth week of NaF treatment. In conclusion, intake of NaF produces alterations in the erythrocyte of the male rat, which indicates induction of oxidative stress. PMID:20640162

Computational Steroidogenesis Model To Predict Biochemical Responses to Endocrine Active Chemicals: Model Development and Cross Validation

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme-mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine active chem...

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

The Role of Cholesterol Utilization in a Computational Adrenal Steroidogenesis Model to Improve Predictability of Biochemical Responses to Endocrine Active Chemicals

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme-mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine active chem...

Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

PubMed

Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

2016-08-01

The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. © 2016 Wiley Periodicals, Inc.

Mechanistic insights into the regulation of metabolic enzymes by acetylation

PubMed Central

2012-01-01

The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour.

PubMed

Mathy, Nathalie; Hébert, Agnès; Mervelet, Peggy; Bénard, Lionel; Dorléans, Audrey; Li de la Sierra-Gallay, Inés; Noirot, Philippe; Putzer, Harald; Condon, Ciarán

2010-01-01

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5'-to-3' exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5'-to-3' exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

Effect of N-benzoyl-D-phenylalanine and metformin on carbohydrate metabolic enzymes in neonatal streptozotocin diabetic rats.

PubMed

Ashokkumar, Natarajan; Pari, Leelavinothan

2005-01-01

The effect of N-benzoyl-D-phenylalanine (NBDP) and metformin was studied on the activities of carbohydrate metabolic enzymes in neonatal streptozotocin (nSTZ) non-insulin-dependent diabetic rats. To induce non-insulin-dependent diabetes mellitus (NIDDM), single dose injection of streptozotocin (STZ; 100 mg/kg body weight; i.p.) was given to 2-day old rats. After 10-12 weeks, rats weighing >150 g were selected for screening in NIDDM model, they were checked for fasting blood glucose concentrations to conform the status of NIDDM. NBDP (50,100 and 200 mg/kg body weight) was administered orally for 6 weeks into the confirmed diabetic rats. The activities of gluconeogenic enzymes were significantly increased, whereas the activities of hexokinase and glucose-6-phosphate dehydrogenase were significantly decreased in nSTZ diabetic rats. Both NBDP and metformin were able to restore the altered enzyme activities to almost control concentrations. Combination treatment was more effective than either drug alone. The administration of NBDP along with metformin to nSTZ diabetic rats normalizes blood glucose and causes marked improvement of altered carbohydrate metabolic enzymes during diabetes.

[Protein fractions and their enzyme activity in the rat myocardium in a Kosmos-936 biosatellite experiment].

PubMed

Tigranian, R A; Nosova, E A; Kolchina, E V; Veresotskaia, N A; Kurkina, L M

1981-01-01

The effect of artificial gravity on protein fractions and their enzyme activity in the myocardium of rats flown on board Cosmos-936 was studied. In weightless rats the content of sarcoplasmic proteins increased at R + O and that of T fraction proteins decreased at R + 25. In centrifuged rats such changes were not seen. In centrifuged rats the enzyme activity of sarcoplasmic proteins did not alter. In weightless rats ATPase activity of myosin decreased significantly, and in centrifuged rats it remained almost unchanged.

DNA in a Tunnel: A Comfy Spot for Recognition - or -The Structure of BsoBI Complexed with DNA. What can we Learn about Function via Structure Determination and how can this be Applied to Bone or Muscle Biology?

NASA Technical Reports Server (NTRS)

vanderWoerd, Mark

2004-01-01

The structure and function of a biologically active molecule are related. To understand its function, it is necessary (but not always sufficient) to know the structure of the molecule. There are many ways of relating the molecular function with the structure. Mutation analysis can identify pertinent amino acids of an enzyme, or alternatively structure comparison of the of two similar molecules with different function may lead to understanding which parts are responsible for a functional aspect, or a series of "structural cartoons" - enzyme structure, enzyme plus substrate, enzyme with transition state analog, and enzyme with product - may give insight in the function of a molecule. As an example we will discuss the structure and function of the restriction enzyme BsoBI from Bacillus stearothemzophilus in complex with its cognate DNA. The enzyme forms a unique complex with DNA in that it completely encircles the DNA. The structure reveals the enzyme-DNA contacts, how the DNA is distorted compared with the canonical forms, and elegantly shows how two distinct DNA sequences can be recognized with the same efficiency. Based on the structure we may also propose a hypothesis how the enzymatic mechanism works. The knowledge gained thru studies such as this one can be used to alter the function by changing the molecular structure. Usually this is done by design of inhibitors specifically active against and fitting into an active site of the enzyme of choice. In the case of BsoBI one of the objectives of the study was to alter the enzyme specificity. In bone biology there are many candidates available for molecular study in order to explain, alter, or (temporarily) suspend activity. For example, the understanding of a pathway that negatively regulates bone formation may be a good target for drug design to stimulate bone formation and have good potential as the basis for new countermeasures against bone loss. In principle the same approach may aid muscle atrophy, radiation damage, immune response changes and other risks identified for long-duration Space travel.

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

PubMed

Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

2015-04-21

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, we demonstrate the key role of MD in combination with crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of which is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as well as the accessibility of water to different regions of it. Each of these examples reveals something different about how mutations can influence enzyme activity and shows that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ways.

Phenylethynyl-butyltellurium inhibits the sulfhydryl enzyme Na+, K+ -ATPase: an effect dependent on the tellurium atom.

PubMed

Quines, Caroline B; Rosa, Suzan G; Neto, José S S; Zeni, Gilson; Nogueira, Cristina W

2013-11-01

Organotellurium compounds are known for their toxicological effects. These effects may be associated with the chemical structure of these compounds and the oxidation state of the tellurium atom. In this context, 2-phenylethynyl-butyltellurium (PEBT) inhibits the activity of the sulfhydryl enzyme, δ-aminolevulinate dehydratase. The present study investigated on the importance of the tellurium atom in the PEBT ability to oxidize mono- and dithiols of low molecular weight and sulfhydryl enzymes in vitro. PEBT, at high micromolar concentrations, oxidized dithiothreitol (DTT) and inhibited cerebral Na(+), K(+)-ATPase activity, but did not alter the lactate dehydrogenase activity. The inhibition of cerebral Na(+), K(+)-ATPase activity was completely restored by DTT. By contrast, 2-phenylethynyl-butyl, a molecule without the tellurium atom, neither oxidized DTT nor altered the Na(+), K(+)-ATPase activity. In conclusion, the tellurium atom of PEBT is crucial for the catalytic oxidation of sulfhydryl groups from thiols of low molecular weight and from Na(+), K(+)-ATPase.

Influence of Background Genome on Enzymatic Characteristics of Yellow (Ay/-, Avy/-) Mice

PubMed Central

Wolff, George L.; Pitot, Henry C.

1973-01-01

Identification of the fundamental polypeptide difference between yellow (Ay/-, Avy/-) and non-yellow mice is important for biomedical research because of the influence of the yellow genotype on normal and neoplastic growth and obesity. The complexity of the "yellow mouse syndrome" makes attainment of this objective dependent on the separation of those pleiotropic enzyme differences which are secondary, and depend on the background genome, from those which are primary, and depend primarily on the agouti locus genotype.—Four of nine hepatic enzyme activities assayed simultaneously differed between eight-week-old yellow (Ay/-, Avy/-) and non-yellow (A/-, a/a) male inbred and F1 hybrid mice. Among these four, only cytoplasmic malic enzyme activity was elevated in all yellow mice, as compared with the non-yellow sibs, regardless of background genome. Glucokinase, serine dehydratase, and tyrosine α-ketoglutarate transaminase activities were also changed in yellow mice, but these alterations depended on the background genome.—The ratio of malic enzyme activity to citrate-cleavage enzyme activity, possibly related to the altered fat metabolism of yellow mice, was influenced by background genome as well as by the yellow genotype.——Significant deviations of enzyme activities from mid-parent values among F1 hybrids were associated with particular background genomes; the number of such deviations was larger among yellow mice than among non-yellows and this difference was greater among C3H F1 hybrids than among C57BL/6 F1 hybrids. PMID:4405752

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes.

PubMed

Oreščanin-Dušić, Zorana; Tatalović, Nikola; Vidonja-Uzelac, Teodora; Nestorov, Jelena; Nikolić-Kokić, Aleksandra; Mijušković, Ana; Spasić, Mihajlo; Paškulin, Roman; Bresjanac, Mara; Blagojević, Duško

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga . It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H 2 O 2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H 2 O 2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

PubMed Central

Paškulin, Roman

2018-01-01

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis. PMID:29599898

Purification and properties of dihydrofolate reductase from cultured mammalian cells

PubMed Central

Gauldie, Jack; Marshall, Lyse; Hillcoat, Brian L.

1973-01-01

Dihydrofolate reductase was purified quickly and simply from small quantities of cultured mammalian cells by affinity chromatography. On gel electrophoresis of the purified enzyme, multiple bands of activity resulted from enzyme–buffer interaction at low but not high buffer concentration. A Ferguson plot (Ferguson, 1964) showed that this heterogeneity was due to a charge difference with no alteration in the size of the enzyme. Stimulation of enzyme activity by KCl, urea and p-hydroxymercuribenzoate, and inhibition by methotrexate and trimethoprim, showed only minor differences between the various enzymes. PMID:4723779

Drug metabolism alterations in nonalcoholic fatty liver disease

PubMed Central

Merrell, Matthew D.; Cherrington, Nathan J.

2013-01-01

Drug-metabolizing enzymes play a vital role in the elimination of the majority of therapeutic drugs. The major organ involved in drug metabolism is the liver. Chronic liver diseases have been identified as a potential source of significant interindividual variation in metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States, affecting between 60 and 90 million Americans, yet the vast majority of NAFLD patients are undiagnosed. NAFLD encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis and fibrosis. Numerous animal studies have investigated the effects of NAFLD on hepatic gene expression, observing significant alterations in mRNA, protein, and activity levels. Information on the effects of NAFLD in human patients is limited, though several significant investigations have recently been published. Significant alterations in the activity of drug-metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions. With the enormous prevalence of NAFLD, it is conceivable that every drug currently on the market is being given to patients with NAFLD. The current review is intended to present the results from both animal models and human patients, summarizing the observed alterations in the expression and activity of the phase I and II drug-metabolizing enzymes. PMID:21612324

Genetic alterations in Krebs cycle and its impact on cancer pathogenesis.

PubMed

Sajnani, Karishma; Islam, Farhadul; Smith, Robert Anthony; Gopalan, Vinod; Lam, Alfred King-Yin

2017-04-01

Cancer cells exhibit alterations in many cellular processes, including oxygen sensing and energy metabolism. Glycolysis in non-oxygen condition is the main energy production process in cancer rather than mitochondrial respiration as in benign cells. Genetic and epigenetic alterations of Krebs cycle enzymes favour the shift of cancer cells from oxidative phosphorylation to anaerobic glycolysis. Mutations in genes encoding aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, and citrate synthase are noted in many cancers. Abnormalities of Krebs cycle enzymes cause ectopic production of Krebs cycle intermediates (oncometabolites) such as 2-hydroxyglutarate, and citrate. These oncometabolites stabilize hypoxia inducible factor 1 (HIF1), nuclear factor like 2 (Nrf2), inhibit p53 and prolyl hydroxylase 3 (PDH3) activities as well as regulate DNA/histone methylation, which in turn activate cell growth signalling. They also stimulate increased glutaminolysis, glycolysis and production of reactive oxygen species (ROS). Additionally, genetic alterations in Krebs cycle enzymes are involved with increased fatty acid β-oxidations and epithelial mesenchymal transition (EMT) induction. These altered phenomena in cancer could in turn promote carcinogenesis by stimulating cell proliferation and survival. Overall, epigenetic and genetic changes of Krebs cycle enzymes lead to the production of oncometabolite intermediates, which are important driving forces of cancer pathogenesis and progression. Understanding and applying the knowledge of these mechanisms opens new therapeutic options for patients with cancer. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Effect of ionic liquid on activity, stability, and structure of enzymes: a review.

PubMed

Naushad, Mu; Alothman, Zied Abdullah; Khan, Abbul Bashar; Ali, Maroof

2012-11-01

Ionic liquids have shown their potential as a solvent media for many enzymatic reactions as well as protein preservation, because of their unusual characteristics. It is also observed that change in cation or anion alters the physiochemical properties of the ionic liquids, which in turn influence the enzymatic reactions by altering the structure, activity, enatioselectivity, and stability of the enzymes. Thus, it is utmost need of the researchers to have full understanding of these influences created by ionic liquids before choosing or developing an ionic liquid to serve as solvent media for enzymatic reaction or protein preservation. So, in the present review, we try to shed light on effects of ionic liquids chemistry on structure, stability, and activity of enzymes, which will be helpful for the researchers in various biocatalytic applications. Copyright © 2012. Published by Elsevier B.V.

Alterations of peptide metabolism and neuropeptidase activity in senile dementia of the Alzheimer's type.

PubMed

Waters, S M; Davis, T P

1997-04-24

Work in our laboratory has shown that in addition to previously characterized changes in the level of neuropeptides in SDAT brain, the activity of degradative enzymes responsible for peptide metabolism is also affected. In addition to other reported alterations in peptide metabolism, we have observed that SS-28 degradation is increased in Brodmann area 22 whereas substance P degradation is increased in temporal cortex. Changes in the degradation of these neuropeptides known to be affected in SDAT correlate well with alterations in the activity of specific neuropeptidases. Trypsin-like serine protease activity is increased in SDAT Brodmann area 22 which parallels the increased degradation of SS-28. The activity of MEP 24.15 is decreased in temporal cortex which corresponds to the decreased degradation of substance P. Changes in the activity of these degradative enzymes in SDAT brain can potentially affect the action of other neuropeptide substrates because the neuropeptidases discussed here terminate the action of several neuropeptides. As more neuropeptide and degradative peptidase alterations are discovered in SDAT, greater emphasis may be placed on the role that peptides and neuropeptidases play in the progression of SDAT.

THE ALTERATION OF INTRACELLULAR ENZYMES

PubMed Central

Kaplan, J. Gordin

1954-01-01

1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C1) 41 ± 3. 2. Above C8 the altering activity of alcohols ceased to follow Traube's rule, and at C18 was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C9 and C12 where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C8, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C1 to C8; aldehydes C1, C2), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially activated by concentrations of altering agent which cause no decrease in viability at all. Hence alteration, unlike death, may not be all-or-none per cell. 6. The fact that the biological criterion being examined was the activation of a water-soluble enzyme rules out the possibility that the reason for the logarithmic increase in altering activity with chain length was increase in concentration of the altering agent in some intracellular fat phase. It is concluded that these surface-active agents cause enzyme alteration by becoming adsorbed at some intracellular interface and thus causing, directly or indirectly, the modification of catalase properties. 7. It is considered that these data support, but do not provide critical proof for, the interfacial hypothesis, which states that catalase is present at the intracellular interface in question, but is desorbed into solution as a consequence of the alteration process. PMID:13211996

Preparation and characterization of a dextran-amylase conjugate.

PubMed

Marshall, J J

1976-07-01

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.

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.

Alterations in kidney enzyme pattern in acute hypervitaminosis A.

PubMed

Alarcón, O M; Reinosa Fuller, J; García de Méndez, G; Agudelo, R; Carnevalí de Tatá, E; Silva, T

1998-06-01

The relation of excessive doses of vitamin A with various kidney pathologies is well known however, information concerning the relation of kidney enzyme activity with acute hypervitaminosis A is rather scarce. In this study we describe the kidney enzymatic alterations observed in rats that received daily intramuscular injections of 10,000, 30,000, 50,000 and 100,000 IU of vitamin A palmitate (VA) during seven days (TREATED GROUPS). A comparison is made with the enzyme activity in healthy rats pair-fed and treated with sodium palmitate by intramuscular injection (CONTROL GROUP). The treated rats showed a proportional increase (p < 0.05) in activity of acid maltase, transminases or aminotransferases (GOT and GPT), alkaline phosphatase (ALP) and acid protease with all doses of VA administered. Amylase, lipase and arginase tend to decrease (p < 0.05) in activity only with doses of 50,000 and 100,000 I.U. of VA. Several factors are responsible for these findings, such as kidney necrosis due to release of lysosomal acid hydrolases produced by hypervitaminosis A.

Solvent effects on enzymes - Implications for extraterrestrial life.

NASA Technical Reports Server (NTRS)

Heinrich, M. R.

1972-01-01

Review of several studies on the alterations taking place in the structure, catalytic activity, specificity, and stability of an enzyme when some or all of the water in the medium is replaced by another solvent. These studies show the utility of solvents as a tool for probing enzyme function. They also suggest that solvents other than water should be investigated as media for controlling and directing enzyme reactions.

Toward reducing immunogenicity of enzyme replacement therapy: altering the specificity of human β-glucuronidase to compensate for α-iduronidase deficiency.

PubMed

Chuang, Huai-Yao; Suen, Ching-Shu; Hwang, Ming-Jing; Roffler, Steve R

2015-11-01

Enzyme replacement therapy (ERT) is an effective treatment for many patients with lysosomal storage disorders caused by deficiency in enzymes involved in cell metabolism. However, immune responses that develop against the administered enzyme in some patients can hinder therapeutic efficacy and cause serious side effects. Here we investigated the feasibility of a general approach to decrease ERT immunogenicity by altering the specificity of a normal endogenous enzyme to compensate for a defective enzyme. We sought to identify human β-glucuronidase variants that display α-iduronidase activity, which is defective in mucopolysaccharidosis (MPS) type I patients. A human β-glucuronidase library was screened by the Enzyme Cleavable Surface-Tethered All-purpose Screen sYstem to isolate variants that exhibited 100-290-fold greater activity against the α-iduronidase substrate 4-methylumbelliferyl α-l-iduronide and 7900-24 500-fold enzymatic specificity shift when compared with wild-type β-glucuronidase. In vitro treatment of MPS I cells with the β-glucuronidase variants significantly restored lysosome appearance similar to treatment with α-iduronidase. Our study suggests that β-glucuronidase variants can be isolated to display α-iduronidase activity and produce significant phenotype improvement of MPS I cells. This strategy may represent a possible approach to produce enzymes that display therapeutic benefits with potentially less immunogenicity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Modification of enzymes by use of high-pressure homogenization.

PubMed

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

Soil enzyme activities in Pinus tabuliformis (Carriere) plantations in northern China

Treesearch

Weiwei Wang; Deborah Page-Dumroese; Ruiheng Lv; Chen Xiao; Guolei Li; Yong Liu

2016-01-01

Changes in forest stand structure may alter the activity of invertase, urease, catalase and phenol oxidase after thinning Pinus tabuliformis (Carriére) plantations in Yanqing County of Beijing, China. We examined changes in these soil enzymes as influenced by time since thinning (24, 32, and 40 years since thinning) for 3 seasons (spring, summer and autumn)...

Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki.

PubMed

Nunes, B; Carvalho, F; Guilhermino, L

2004-12-01

The objective of this study was to investigate both acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of the mosquitofish (Gambusia holbrooki). AChE, commonly used as a biomarker of neurotoxicity, was determined in the total head. LDH, an important enzyme of anaerobic metabolism, was quantified in dorsal muscle, and CAT, enzyme which has been used as indicative parameter of peroxisome proliferation, was determined in the liver. Furthermore, alterations of body and liver weight were also determined, through the calculation of the ratios final body weight/initial body weight, liver weight/final body weight, liver weight/gills weight and liver weight/head weight. Acute exposure of G. holbrooki to both clofibrate and clofibric acid induced a decrease in liver CAT activity, an increase in muscle LDH activity, while no effects were observed on AChE activity. However, chronic exposure did not alter significantly the enzymatic activities, suggesting reduced or null effects over these pathways, relative to effects reported in other species. No effects were observed for the calculated ratios, except a significant weight reduction for males chronically exposed to clofibrate.

The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome

PubMed Central

Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

2016-01-01

During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway. PMID:27335086

High-Fat Diet and Voluntary Chronic Aerobic Exercise Recover Altered Levels of Aging-Related Tryptophan Metabolites along the Kynurenine Pathway

PubMed Central

Lee, Keon-Joo; Cho, Joo-Youn; Lee, Soon-Tae; Kim, Hwa Suk; Shim, Jun Hwa; Lee, Sang Kun; Kim, Manho

2017-01-01

Tryptophan metabolites regulate a variety of physiological processes, and their downstream metabolites enter the kynurenine pathway. Age-related changes of metabolites and activities of associated enzymes in this pathway are suggestable and would be potential intervention targets. Blood levels of serum tryptophan metabolites in C57BL/6 mice of different ages, ranging from 6 weeks to 10 months, were assessed using high-performance liquid chromatography, and the enzyme activities for each metabolic step were estimated using the ratio of appropriate metabolite levels. Mice were subjected to voluntary chronic aerobic exercise or high-fat diet to assess their ability to rescue age-related alterations in the kynurenine pathway. The ratio of serum kynurenic acid (KYNA) to 3-hydroxylkynurenine (3-HK) decreased with advancing age. Voluntary chronic aerobic exercise and high-fat diet rescued the decreased KYNA/3-HK ratio in the 6-month-old and 8-month-old mice groups. Tryptophan metabolites and their associated enzyme activities were significantly altered during aging, and the KYNA/3-HK ratio was a meaningful indicator of aging. Exercise and high-fat diet could potentially recover the reduction of the KYNA/3-HK ratio in the elderly. PMID:28680298

Altered fibre types in gastrocnemius muscle of high wheel-running selected mice with mini-muscle phenotypes.

PubMed

Guderley, Helga; Joanisse, Denis R; Mokas, Sophie; Bilodeau, Geneviève M; Garland, Theodore

2008-03-01

Selective breeding of mice for high voluntary wheel running has favoured characteristics that facilitate sustained, aerobically supported activity, including a "mini-muscle" phenotype with markedly reduced hind limb muscle mass, increased mass-specific activities of oxidative enzymes, decreased % myosin heavy chain IIb, and, in the medial gastrocnemius, reduced twitch speed, reduced mass-specific isotonic power, and increased fatigue resistance. To evaluate whether selection has altered fibre type expression in mice with either "mini" or normal muscle phenotypes, we examined fibre types of red and white gastrocnemius. In both the medial and lateral gastrocnemius, the mini-phenotype increased activities of oxidative enzymes and decreased activities of glycolytic enzymes. In red muscle samples, the mini-phenotype markedly changed fibre types, with the % type I and type IIA fibres and the surface area of type IIA fibres increasing; in addition, mice from selected lines in general had an increased % type IIA fibres and larger type I fibres as compared with mice from control lines. White muscle samples from mini-mice showed dramatic structural alterations, with an atypical distribution of extremely small, unidentifiable fibres surrounded by larger, more oxidative fibres than normally present in white muscle. The increased proportion of oxidative fibres and these atypical small fibres together may explain the reduced mass and increased mitochondrial enzyme activities in mini-muscles. These and previous results demonstrate that extension of selective breeding beyond the time when the response of the selected trait (i.e. distance run) has levelled off can still modify the mechanistic underpinnings of this behaviour.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

PubMed

Huston, Scott; Collins, John; Sun, Fangfang; Zhang, Ting; Vaden, Timothy D; Zhang, Y-H Percival; Fu, Jinglin

2018-05-01

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill

PubMed Central

Kamalanathan, Manoj; Xu, Chen; Schwehr, Kathy; Bretherton, Laura; Beaver, Morgan; Doyle, Shawn M.; Genzer, Jennifer; Hillhouse, Jessica; Sylvan, Jason B.; Santschi, Peter; Quigg, Antonietta

2018-01-01

Extracellular enzymes and extracellular polymeric substances (EPS) play a key role in overall microbial activity, growth and survival in the ocean. EPS, being amphiphilic in nature, can act as biological surfactant in an oil spill situation. Extracellular enzymes help microbes to digest and utilize fractions of organic matter, including EPS, which can stimulate growth and enhance microbial activity. These natural processes might have been altered during the 2010 Deepwater Horizon oil spill due to the presence of hydrocarbon and dispersant. This study aims to investigate the role of bacterial extracellular enzymes during exposure to hydrocarbons and dispersant. Mesocosm studies were conducted using a water accommodated fraction of oil mixed with the chemical dispersant, Corexit (CEWAF) in seawater collected from two different locations in the Gulf of Mexico and corresponding controls (no additions). Activities of five extracellular enzymes typically found in the EPS secreted by the microbial community – α- and β-glucosidase, lipase, alkaline phosphatase, leucine amino-peptidase – were measured using fluorogenic substrates in three different layers of the mesocosm tanks (surface, water column and bottom). Enhanced EPS production and extracellular enzyme activities were observed in the CEWAF treatment compared to the Control. Higher bacterial and micro-aggregate counts were also observed in the CEWAF treatment compared to Controls. Bacterial genera in the order Alteromonadaceae were the most abundant bacterial 16S rRNA amplicons recovered. Genomes of Alteromonadaceae commonly have alkaline phosphatase and leucine aminopeptidase, therefore they may contribute significantly to the measured enzyme activities. Only Alteromonadaceae and Pseudomonadaceae among bacteria detected here have higher percentage of genes for lipase. Piscirickettsiaceae was abundant; genomes from this order commonly have genes for leucine aminopeptidase. Overall, this study provides insights into the alteration to the microbial processes such as EPS and extracellular enzyme production, and to the microbial community, when exposed to the mixture of oil and dispersant. PMID:29740422

Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

PubMed

Lipscomb, William N; Kantrowitz, Evan R

2012-03-20

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

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 resilient even after five years of drought.

Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes.

PubMed

Peng, Lin; Qiao, Shuangkui; Xu, Zhenghong; Guan, Feng; Ding, Zhongyang; Gu, Zhenghua; Zhang, Liang; Shi, Guiyang

2015-11-20

We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reversal of high fat diet-induced obesity through modulating lipid metabolic enzymes and inflammatory markers expressions in rats.

PubMed

A, Kalaivani; Uddandrao, V V Sathibabu; Parim, Brahmanaidu; Ganapathy, Saravanan; P R, Nivedha; Kancharla, Sushma Chandulee; P, Rameshreddy; K, Swapna; Sasikumar, Vadivukkarasi

2018-03-19

In this study, we evaluated the ameliorative potential of Cucurbita maxima seeds oil (CSO (100 mg/kg body weight)) supplementation to high fat diet (HFD)-induced obese rats for 30 days on the changes in body weight, markers of lipid metabolism such as LDL, HDL, triglycerides, total cholesterol, adiponectin, leptin, amylase, and lipase. We also investigated the effects of CSO on the changes of lipid metabolic enzymes such as fatty-acid synthase, acetyl CoA carboxylase, carnitine palmitoyl transferase-1, HMG CoA reductase, and inflammatory markers (TNF-α and IL-6). Administration of CSO revealed significant diminution in body weight gain, altered the activity, expressions of lipid marker enzymes and inflammatory markers. It demonstrated that CSO had considerably altered these parameters when evaluated with HFD control rats. In conclusion, this study suggested that CSO might ameliorate the HFD-induced obesity by altering the enzymes and mRNA expressions important to lipid metabolism.

Alteration of paraoxonase, arylesterase and lactonase activities in people around fluoride endemic area of Tamil Nadu, India.

PubMed

Arulkumar, Mani; Vijayan, Raji; Penislusshiyan, Sakayanathan; Sathishkumar, Palanivel; Angayarkanni, Jayaraman; Palvannan, Thayumanavan

2017-08-01

Toxicity due to excess fluoride concentration in drinking water is of great concern in people who rely only on the ground water as their water source in many region of the world. We collected samples and examined the toxicity of fluoride in a population residing at Salem, Dharmapuri and Krishnagiri districts of Tamil Nadu, India and measured HDL bound enzyme (PON1), erythrocyte membrane bound enzymes (acetylcholinesterase, AChE) and adenosine 5' triphosphatase (ATPases), plasma enzyme (butyrylcholinesterase, BChE) and rate limiting enzyme in heme biosynthesis (delta aminolevulinic acid dehydratase, δ-ALAD) activities. In fluorosis patients, formation of lipid peroxidation product was more in erythrocytes than in plasma. The observation further revealed that there was 50% reduction in the activity of HDL bound anti atherogenic enzyme-paraoxonase (PON1). The activities of membrane bound and signaling enzymes (acetylcholinesterase - AChE and adenosine 5' triphosphatase - ATPase) of erythrocyte were also diminished. These results suggested that there was defectiveness in the signaling and energy metabolism in fluorosis patients. Altered isoenzyme pattern of lactate dehydrogenase (LDH) in fluorosis samples was observed. Furthermore, the result suggested that both the heart (LDH 1) and liver (LDH 5) were most affected by fluoride toxicity. The study also provided reference values for tests which are used to predict the severity of fluoride toxicity. The toxic effect of fluoride was due to the collective effects on vital protective system rather than single factor. Copyright © 2017 Elsevier B.V. All rights reserved.

Function and biotechnology of extremophilic enzymes in low water activity

PubMed Central

2012-01-01

Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

Endophytic fungus Phomopsis liquidambari and different doses of N-fertilizer alter microbial community structure and function in rhizosphere of rice

PubMed Central

Siddikee, Md. Ashaduzzaman; Zereen, Mst Israt; Li, Cai-Feng; Dai, Chuan-Chao

2016-01-01

Microbial community structure and functions of rhizosphere soil of rice were investigated after applying low and high doses of nitrogenous fertilizer and Phomopsis liquidambari. Average well color development, substrate richness, catabolic diversity and soil enzymes activities varied after applying N-fertilizer and P. liquidambari and were greater in P. liquidambari treated soil than only N-fertilization. Multivariate analysis distinctly separated the catabolic and enzymes activity profile which statistically proved alteration of microbial functional diversity. Nitrogen fertilizer altered microbial community structure revealed by the increased content of total PLFAs, specific subgroup marker PLFAs except fungal PLFAs and by the decreased ratio of G+/G−, sat/monunsat, iso/anteiso, F/B except trans/cis while P. liquidambari inoculation enhanced N-fertilization effect except increased fungal PLFA and decreased trans/cis. PCA using identified marker PLFAs revealed definite discrimination among the treatments which further statistically confirmed structural changed of microbial community. Nitrogenase activity representative of N-fixing community decreased in N-fertilizer treatment while P. liquidambari inoculation increased. In short, application of P. liquidambari with low doses of N-fertilizer improved rice growth and reduced N-fertilizer requirement by increasing enzymes activities involved in C, N and P cycling, structural and functional diversity of microbes, nitrogenase activity involved in N2 fixation and accumulation of total-N. PMID:27596935

Computational Model of Adrenal Steroidogenesis to Predict Biochemical Response to Endocrine Disruptors

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme mediated reactions. The activity of steroidogenic enzymes can be altered by various endocrine disrupters (ED), ...

Regular exercise training reverses ectonucleotidase alterations and reduces hyperaggregation of platelets in metabolic syndrome patients.

PubMed

Martins, Caroline Curry; Bagatini, Margarete Dulce; Cardoso, Andréia Machado; Zanini, Daniela; Abdalla, Fátima Husein; Baldissarelli, Jucimara; Dalenogare, Diéssica Padilha; Farinha, Juliano Boufleur; Schetinger, Maria Rosa Chitolina; Morsch, Vera Maria

2016-02-15

Alterations in the activity of ectonucleotidase enzymes have been implicated in cardiovascular diseases, whereas regular exercise training has been shown to prevent these alterations. However, nothing is known about it relating to metabolic syndrome (MetS). We investigated the effect of exercise training on platelet ectonucleotidase enzymes and on the aggregation profile of MetS patients. We studied 38 MetS patients who performed regular concurrent exercise training for 30 weeks. Anthropometric measurements, biochemical profiles, hydrolysis of adenine nucleotides in platelets and platelet aggregation were collected from patients before and after the exercise intervention as well as from individuals of the control group. An increase in the hydrolysis of adenine nucleotides (ATP, ADP and AMP) and a decrease in adenosine deamination in the platelets of MetS patients before the exercise intervention were observed (P<0.001). However, these alterations were reversed by exercise training (P<0.001). Additionally, an increase in platelet aggregation was observed in the MetS patients (P<0.001) and the exercise training prevented platelet hyperaggregation in addition to decrease the classic cardiovascular risks. An alteration of ectonucleotidase enzymes occurs during MetS, whereas regular exercise training had a protective effect on these enzymes and on platelet aggregation. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Response of the antioxidant enzymes of the erythrocyte and alterations in the serum biomarkers in rats following oral administration of nanoparticles.

PubMed

Canli, Esin G; Atli, Gülüzar; Canli, Mustafa

2017-03-01

In this study, Al 2 O 3 , CuO and TiO 2 nanoparticles (NPs) were administered to mature female rats (Rattus norvegicus var. albinos) via oral gavage (0, 0.5, 5, 50mg/kg b.w./day) for 14days to investigate their effects on 14 serum biomarkers and 4 antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase) activities in the erythrocyte. Data showed that Al 2 O 3 did not cause any significant (P>0.05) change in the parameters, except few cases, while CuO and TiO 2 caused significant alterations in antioxidant system parameters of the erythrocytes. Activities of catalase and superoxide dismutase significantly decreased in CuO and TiO 2 administered rats. Oppositely, glutathione peroxidase activity increased in CuO and TiO 2 administered rats. There were no significant alterations in the activity of glutathione S-transferase in the erythrocytes. Levels of glucose, cholesterol, bilirubin, triglyceride, triiodothyronine (T3), estradiol, prolactin and immunoglobulin M (IgM) in the serum altered after some of NP administrations, whereas cortisol, protein, creatinine, blood urea nitrogen (BUN), thyroxine (T4) and immunoglobulin G (IgG) levels in the serum did not change significantly after any of NP administration. There were outstanding increases in the levels of bilirubin and prolactin and decreases in the levels of triglyceride and estradiol. The present study demonstrated that the antioxidant enzymes in the erythrocyte were generally affected from copper and titanium NPs, while aluminium and copper NPs caused more significant alterations in serum biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

PubMed Central

Piao, Yun-shang; Wiesenfeld, Paddy; Sprando, Robert; Arnold, Julia T.

2013-01-01

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFβ1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFβ1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFβ1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFβ1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3β hydroxysteroid dehydrogenase (HSD), and down-regulation of 17βHSD5, and 17βHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens. PMID:23770322

TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

PubMed

Piao, Yun-shang; Wiesenfeld, Paddy; Sprando, Robert; Arnold, Julia T

2013-11-01

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFβ1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFβ1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFβ1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFβ1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3β hydroxysteroid dehydrogenase (HSD), and down-regulation of 17βHSD5, and 17βHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens. Published by Elsevier Ltd.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

Elevated enzyme activities in soils under the invasive nitrogen-fixing tree Falcataria moluccana

Treesearch

Steven D. Allison; Caroline Nielsen; R. Flint Hughes

2006-01-01

Like other N-fixing invasive species in Hawaii, Falcataria moluccana dramatically alters forest structure, litterfall quality and quantity, and nutrient dynamics. We hypothesized that these biogeochemical changes would also affect the soil microbial community and the extracellular enzymes responsible for carbon and nutrient mineralization. Across...

Autolytic defective mutant of Streptococcus faecalis.

PubMed Central

Cornett, J B; Redman, B E; Shockman, G D

1978-01-01

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme. PMID:415045

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

PubMed

Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

2015-07-15

Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

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

Warming rate drives microbial limitation and enzyme expression during peat decomposition

NASA Astrophysics Data System (ADS)

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

2015-12-01

Recent developments of enzyme-based decomposition models highlight the importance of enzyme kinetics with warming, but most modeling exercises are based on studies with a step-wise warming. This approach may mask the effect of temperature in controlling in-situ activities as in most ecosystems soil temperature change more gradually than air temperature. We conducted an experiment to test the effects of contrasting warming rates on the kinetics of C, N, and P degradation enzymes in subtropical peat soils. We also wanted to evaluate if the stoichiometry of enzyme kinetics shifts under contrasting warming rates and if so, how does it relate to the stoichiometry in microbial biomass. Contrasting warming rates altered microbial biomass stoichiometry leading to differing patterns of enzyme expression and microbial nutrient limitation. Activity (higher Vmax) and efficiency (lower Km) of C acquisition enzymes were greater in the step treatment; however, expressions of nutrient (N and P) acquiring enzymes were enhanced in the ramp treatment at the end of the experiment. In the step treatment, there was a typical pattern of an initial peak in the Vmax and drop in the Km for all enzyme groups followed by later adjustments. On the other hand, a consistent increase in Vmax and decline in Km of all enzyme groups were observed in the slow warming treatment. These changes were sufficient to alter microbial identity (as indicated by enzyme Km and biomass stoichiometry) with two apparently stable endpoints under contrasting warming rates. This observation resembles the concept of alternate stable states and highlights a need for improved representation of warming in models.

Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1.

PubMed

Hwa, Kuo Yuan; Subramani, Boopathi; Shen, San-Tai; Lee, Yu-May

2015-09-01

β-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with β-glucosidase, β-mannosidase, β-fucosidase and β-galactosidase activities. Sequence alignment with other β-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic β-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its β-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for β-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures. Copyright © 2015 Elsevier Inc. All rights reserved.

d-limonene ameliorates diabetes and its complications in streptozotocin-induced diabetic rats.

PubMed

Bacanlı, Merve; Anlar, Hatice Gül; Aydın, Sevtap; Çal, Tuğbagül; Arı, Nuray; Ündeğer Bucurgat, Ülkü; Başaran, A Ahmet; Başaran, Nurşen

2017-12-01

It is known that diabetes causes some complications including alterations in lipid profile, hepatic enzyme levels but also it causes oxidative stress. Limonene, a major component of Citrus oils, has important health beneficial effects in lowering the level of oxidative stress due to its antioxidant activity. The aim of this study was to investigate the effects of D-limonene on streptozotocin (STZ)-induced diabetes in Wistar albino rats. For this purpose, DNA damage was evaluated by alkaline comet assay. Changes in the activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GSHPx) and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), total glutathione (GSH), malondialdehyde (MDA), insulin, total bilirubin and BCA protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol and triglyceride were also evaluated. D-limonene treatment was found to significantly decrease DNA damage, GR enzyme activities and MDA levels and significantly increase GSH levels and CAT, SOD and GSH-Px enzyme activities and altered lipid and liver enzyme parameters in diabetic rats. According to our results, it seems that D-limonene might have a role in the prevention of the complication of diabetes in rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2015-11-24

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2013-04-09

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Alteration and modulation of protein activity by varying post-translational modification

DOEpatents

Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

2015-03-03

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Alteration and modulation of protein activity by varying post-translational modification

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

Thompson, David N.; Reed, David W.; Thompson, Vicki S.

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

PubMed Central

Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

2013-01-01

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

Experimental neonatal hypoxia ischemia causes long lasting changes of oxidative stress parameters in the hippocampus and the spleen.

PubMed

Odorcyk, Felipe Kawa; Kolling, Janaína; Sanches, Eduardo Farias; Wyse, Angela T S; Netto, Carlos Alexandre

2018-05-24

Neonatal hypoxia ischemia (HI) is the main cause of mortality and morbidity in newborns. The mechanisms involved in its progression start immediately and persist for several days. Oxidative stress and inflammation are determinant factors of the severity of the final lesion. The spleen plays a major part in the inflammatory response to HI. This study assessed the temporal progression of HI-induced alterations in oxidative stress parameters in the hippocampus, the most affected brain structure, and in the spleen. HI was induced in Wistar rat pups in post-natal day 7. Production of reactive oxygen species (ROS), and the activity of the anti oxidant enzyme superoxide dismutase and catalase were assessed 24 h, 96 h and 38 days post-HI. Interestingly, both structures showed a similar pattern, with few alterations in the production of ROS species up to 96 h often combined with an increased activity of the anti oxidant enzymes. However, 38 days after the injury, ROS were at the highest in both structures, coupled with a decrease in the activity of the enzymes. Altogether, present results suggest that HI causes long lasting alterations in the hippocampus as well as in the spleen, suggesting a possible target for delayed treatments for HI.

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

PubMed Central

Bulleid, N J; Graham, A B; Craft, J A

1986-01-01

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms. Images Fig. 2. Fig. 3. PMID:3082328

Glycation of human erythrocyte glutathione peroxidase: effect on the physical and kinetic properties.

PubMed

Suravajjala, Sreekanth; Cohenford, Menashi; Frost, Leslie R; Pampati, Praveen K; Dain, Joel A

2013-06-05

Glutathione peroxidase (GPx) is a significant antioxidant enzyme that plays a key role in protecting the body from reactive oxygen species (ROS) and their toxicity. As a biocatalyst, the enzyme has been shown to reduce hydrogen peroxide to water and lipid hydroperoxides to their respective alcohols. The increased levels of ROS in patients with diabetes have been speculated to arise, in part, from alterations in the activity of glutathione antioxidant enzymes, perhaps, by mechanisms such as the glycation of the protein, in vivo. Under physiological conditions of temperature and pH, we investigated the susceptibility of human glutathione peroxidase to glycation, determined the effects of glycation on the physical and kinetic properties of the enzyme, and identified the protein's vulnerable amino acid sites of glycation. Circular dichroism, UV and mass spectrometry studies revealed that methylglyoxal and DL-glyceraldehyde are potent glycators of glutathione peroxidase; destabilizing its structure, altering its pH activity and stability profiles and increasing its Km value. In comparison to DL-glyceraldehyde, methylglyxol was a more potent glycator of the enzyme and was found to nonenzymatically condense with Arg-177, located near the glutathione binding site of GPx. Copyright © 2013 Elsevier B.V. All rights reserved.

Migratory preparation associated alterations in pectoralis muscle biochemistry and proteome in Palearctic-Indian emberizid migratory finch, red-headed bunting, Emberiza bruniceps.

PubMed

Banerjee, Somanshu; Chaturvedi, Chandra Mohini

2016-03-01

Avian migration is an exceptionally high-energy-demanding process, which is met by the accumulation and utilization of fuel stores as well as the alterations in muscle physiology prior to their flight. Pre-migratory fattening coupled with changes in flight muscle metabolic enzymes and proteome is required to provide the necessary fuel and muscle performance required for migration. We studied how the serum metabolites (urea, uric acid, and creatinine), pectoralis muscle metabolites (glycogen, glucose, and cholesterol), muscle metabolic enzymes (CPT, HOAD, CS, MDH, CCO, CK, LDH, PFK, MLPL, and PK), liver lipogenic enzyme (FAS), and pectoralis muscle proteins get altered in pre-migratory and non-migratory buntings. Significantly increased pectoralis muscle fatty acid oxidation (CPT and HOAD activity), aerobic/anaerobic capacity (CS, CCO, and MDH activity), glycolytic capacity (PFK and PK activity), lipolysis (muscle LPL), and burst power (CK activity) were observed prior to the spring migration in pre-migratory buntings, whereas significantly increased pectoralis muscle anaerobic capacity (LDH activity) was observed in non-migratory buntings. Significant increase in the liver FAS showed profound lipogenesis prior to the spring migration. In this study, we have also investigated whether muscle has differential protein content during the pre-migratory and non-migratory phases of the annual migratory cycle. Twenty-nine proteins are identified and well characterized varying in expression significantly during the pre-migratory and non-migratory phases. These findings indicate that significant pre-migratory fattening and alterations in flight (pectoralis) muscle biochemistry and proteome in between the non- and pre-migratory phases may play a significant role in pre-migratory flight muscle preparation in these long-route migrants. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipase in aqueous-polar organic solvents: Activity, structure, and stability

PubMed Central

Kamal, Md Zahid; Yedavalli, Poornima; Deshmukh, Mandar V; Rao, Nalam Madhusudhana

2013-01-01

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ∼20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]-HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active-site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased Km); however significantly enhanced the kcat. Melting temperature (Tm) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein. PMID:23625694

ALTERED DEVELOPMENT AND REPRODUCTION IN MOSQUITOFISH EXPOSED TO PULP AND PAPER MILL EFFLUENT IN THE FENHOLLOW RIVER, FLORIDA USA

EPA Science Inventory

Female mosquitofish exposed to pulp and paper mill effluent (PME) in the Fenholloway River, Florida, USA have masculinized secondary sex characteristics and altered aromatase enzyme activity. We and others have shown that the Fenholloway River PME contains androgenic and progesto...

Rhododendron thickets alter N Cycling and soul extracellular enzyme activities in southern Appalachian hardwood forests

Treesearch

Nina Wurzburger; Ronald L. Hendrick

2007-01-01

Rhododendron maximum L., a spreading understory shrub, inhibits overstory. Regeneration and alters forest community structure in southern Appalachian hardwood forests. Using paired plots and reciprocal litter transplants in forests with and without R. maximum cover, we examined the influence of R. maximum on Leaf...

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

PubMed

Serrano-Hervás, Eila; Casadevall, Guillem; Garcia-Borràs, Marc; Feixas, Ferran; Osuna, Sílvia

2018-04-06

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

PubMed

Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

2015-01-02

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

Pregnancy-related pharmacokinetic changes.

PubMed

Tasnif, Y; Morado, J; Hebert, M F

2016-07-01

The pharmacokinetics of many drugs are altered by pregnancy. Drug distribution and protein binding are changed by pregnancy. While some drug metabolizing enzymes have an apparent increase in activity, others have an apparent decrease in activity. Not only is drug metabolism affected by pregnancy, but renal filtration is also increased. In addition, pregnancy alters the apparent activities of multiple drug transporters resulting in changes in the net renal secretion of drugs. © 2016 American Society for Clinical Pharmacology and Therapeutics.

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

Acute Exposure of Medaka to Carcinogens: An Ultrastructural, Cytochemical and Morphometric Analysis of Liver and Kidney

DTIC Science & Technology

1993-05-20

statements regarding acid phosphatase activity are not possible because of the inconsistency of labeling. Morphometric analysis were conducted on ratios of...may result from the combining of TCE and other toxic substances. The two enzyme systems selected for examination, acid phosphatase and peroxidase are...administration of DENA have demonstrated altered activity of several enzymes, including acid phosphatase (Fischer et al., 1983). Long term treatment of rodents

Duckweed diversity decreases heavy metal toxicity by altering the metabolic function of associated microbial communities.

PubMed

Zhao, Zhao; Shi, Huijuan; Liu, Cunqi; Kang, Xianjiang; Chen, Lingci; Liang, Xiaofei; Jin, Lei

2018-07-01

Mono-cultured and mix-cultured duckweed species were investigated with respect to the function of their associated microbial communities in heavy metal contaminated wastewater. Results show that the carbon source utilization patterns of the L. aequinoctialis- and S. polyrhiza-associated microbial communities were different. The relationships between microbial activity, antioxidant enzyme activity (CAT, GSH, and SOD) and growth was positive and significant. The microbial activity of L. aequinoctialis and S. polyrhiza in mixture was higher than in monoculture in low and high heavy metal, respectively, thereby altering the utilization of specific carbon source types and increasing duckweed growth and antioxidant enzyme activity, when compared to the monocultured duckweed. Furthermore, results indicate that duckweed species in mixture are protected from damage through regulation of the associated bacterial communities. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

PubMed

Nadar, Shamraja S; Rathod, Virendra K

2017-08-22

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

A significant increase in both basal and maximal calcineurin activity following fluid percussion injury in the rat.

PubMed

Kurz, Jonathan E; Parsons, J Travis; Rana, Aniruddha; Gibson, Cynthia J; Hamm, Robert J; Churn, Severn B

2005-04-01

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Do alterations in follicular fluid proteases contribute to human infertility?

PubMed

Cookingham, Lisa Marii; Van Voorhis, Bradley J; Ascoli, Mario

2015-05-01

Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

FT-Raman spectroscopic analysis of enhanced activity of supercritical carbon dioxide treated bacterial alpha-amylase.

PubMed

Paul, Kaninika; Dutta, Sayantani; Bhattacharjee, Paramita

2017-09-01

Our previous investigation on high pressure supercritical carbon dioxide treatment of a bacterial α-amylase had revealed enhanced activity of the same. 1 H NMR analysis of the activity enhanced enzyme led the authors to hypothesize that the enhancement was possibly owing to alterations in the active site of the enzyme. In the present study, the changes in the active site of the treated enzyme was analysed by Fourier-transform Raman (FT-Raman) spectroscopy. The spectra obtained revealed shifting of bands in the active site of α-amylase indicating a nudging effect of the bonds in this region consequent to high pressure treatment. Also, shifts in bands in the OH stretching vibration of water were observed in the enzyme spectra. These variations in the spectra confirmed changes in the active site as well as in the water associated with the same that perhaps had a concerted effect on the increased activity of α-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

Geraniol modulates tongue and hepatic phase I and phase II conjugation activities and may contribute directly to the chemopreventive activity against experimental oral carcinogenesis.

PubMed

Madankumar, Arumugam; Jayakumar, Subramaniyan; Gokuladhas, Krishnan; Rajan, Balan; Raghunandhakumar, Subramanian; Asokkumar, Selvamani; Devaki, Thiruvengadam

2013-04-05

Xenobiotic metabolizing enzymes are chief determinants in both the susceptibility to mutagenic effect of chemical carcinogens and in the response of tumors to chemotherapy. The present study was aimed to analyze the effect of geraniol administration on the activity of phase I and phase II carcinogen metabolizing enzymes through the nuclear factor erythroid 2-related factor-2 (Nrf2) activation against 4-niroquinoline-1-oxide (4NQO) induced oral carcinogenesis. The well-known chemical carcinogen 4NQO (50 ppm) was used to induce oral carcinogenesis through drinking water for 4, 12, and 20 weeks. The degree of cancer progression at each stage was confirmed by histological examination. At the end of the experimental period, 100% tumor formation was observed in the oral cavity of 4NQO induced animals with significant (P<0.05) alteration in the status of tumor markers, tongue and liver phase I and phase II drug metabolizing enzymes indicating progression of disease. Oral administration of geraniol at the dose of 200 mg/kg b.wt., thrice a week to 4NQO induced animals was able to inhibit tumor formation and thereby delayed the progression of oral carcinogenesis by modulating tongue and liver phase I and phase II drug metabolizing enzymes, as substantiated further by the histological and transmission electron microscopic studies. Our results demonstrate that geraniol exerts its chemopreventive potential by altering activities of phases I and II drug metabolizing enzymes to achieve minimum bioactivation of carcinogen and maximum detoxification. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of the electrical currents generated by the intestinal smooth muscle layers on pancreatic enzyme activity: an in vitro study.

PubMed

Dabek, Marta; Podgurniak, Paweł; Piedra, Jose L Valverde; Szymańczyk, Sylwia; Filip, Rafał; Wojtasz-Pajak, Anna; Werpachowska, Eliza; Podgurniak, Malgorzata; Pierzynowski, Stefan G

2007-05-01

Gut enzymes in the small intestine are exposed to extremely low electrical currents (ELEC) generated by the smooth muscle. In the present study, the in vitro tests were undertaken to evaluate the effect of these electric currents on the activity of the proteolytic pancreatic digestive enzymes. A simulator generating the typical electrical activity of pig gut was used for these studies. The electric current emitted by the simulator was transmitted to the samples, containing enzyme and its substrate, using platinum plate electrodes. All samples were incubated at 37 degrees C for 1 h. The changes in optical density, corresponding to enzyme activity, in samples stimulated for 1 h with ELEC was compared with that not exposed to ELEC. The obtained results show that the electrical current with the characteristics of the myoelectrical migrating complex (MMC) has an influence on pancreatic enzyme activity. Increased endopeptidase and reduced exopeptidase activity was noticed in samples treated with ELEC. This observation can be of important as analyzed factors which can alter enzymatic activity of the gut, can thus also affect feed/food digestibility. (c) 2007 Wiley-Liss, Inc.

Pre-harvest UV-C irradiation triggers VOCs accumulation with alteration of antioxidant enzymes and phytohormones in strawberry leaves.

PubMed

Xu, Yanqun; Luo, Zisheng; Charles, Marie Thérèse; Rolland, Daniel; Roussel, Dominique

2017-11-01

Recent studies have highlighted the biological and physiological effects of pre-harvest ultraviolet (UV)-C treatment on growing plants. However, little is known about the involvement of volatile organic compounds (VOCs) and their response to this treatment. In this study, strawberry plants were exposed to three different doses of UV-C radiation for seven weeks (a low dose: 9.6kJm -2 ; a medium dose: 15kJm -2 ; and a high-dose: 29.4kJm -2 ). Changes in VOC profiles were investigated and an attempt was made to identify factors that may be involved in the regulation of these alterations. Principle compounds analysis revealed that VOC profiles of UV-C treated samples were significantly altered with 26 VOCs being the major contributors to segregation. Among them, 18 fatty acid-derived VOCs accumulated in plants that received high and medium dose of UV-C treatments with higher lipoxygenase and alcohol dehydrogenase activities. In treated samples, the activity of the antioxidant enzymes catalase and peroxidase was inhibited, resulting in a reduced antioxidant capacity and higher lipid peroxidation. Simultaneously, jasmonic acid level was 74% higher in the high-dose group while abscisic acid content was more than 12% lower in both the medium and high-dose UV-C treated samples. These results indicated that pre-harvest UV-C treatment stimulated the biosynthesis of fatty acid-derived VOCs in strawberry leaf tissue by upregulating the activity of enzymes of the LOX biosynthetic pathway and downregulating antioxidant enzyme activities. It is further suggested that the mechanisms underlying fatty acid-derived VOCs biosynthesis in UV-C treated strawberry leaves are associated with UV-C-induced changes in phytohormone profiles. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

Field and lab conditions alter microbial enzyme and biomass dynamics driving decomposition of the same leaf litter

PubMed Central

Rinkes, Zachary L.; Sinsabaugh, Robert L.; Moorhead, Daryl L.; Grandy, A. Stuart; Weintraub, Michael N.

2013-01-01

Fluctuations in climate and edaphic factors influence field decomposition rates and preclude a complete understanding of how microbial communities respond to plant litter quality. In contrast, laboratory microcosms isolate the intrinsic effects of litter chemistry and microbial community from extrinsic effects of environmental variation. Used together, these paired approaches provide mechanistic insights to decomposition processes. In order to elucidate the microbial mechanisms underlying how environmental conditions alter the trajectory of decay, we characterized microbial biomass, respiration, enzyme activities, and nutrient dynamics during early (<10% mass loss), mid- (10–40% mass loss), and late (>40% mass loss) decay in parallel field and laboratory litter bag incubations for deciduous tree litters with varying recalcitrance (dogwood < maple < maple-oak mixture < oak). In the field, mass loss was minimal (<10%) over the first 50 days (January–February), even for labile litter types, despite above-freezing soil temperatures and adequate moisture during these winter months. In contrast, microcosms displayed high C mineralization rates in the first week. During mid-decay, the labile dogwood and maple litters in the field had higher mass loss per unit enzyme activity than the lab, possibly due to leaching of soluble compounds. Microbial biomass to litter mass (B:C) ratios peaked in the field during late decay, but B:C ratios declined between mid- and late decay in the lab. Thus, microbial biomass did not have a consistent relationship with litter quality between studies. Higher oxidative enzyme activities in oak litters in the field, and higher nitrogen (N) accumulation in the lab microcosms occurred in late decay. We speculate that elevated N suppressed fungal activity and/or biomass in microcosms. Our results suggest that differences in microbial biomass and enzyme dynamics alter the decay trajectory of the same leaf litter under field and lab conditions. PMID:24027563

Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887

NASA Technical Reports Server (NTRS)

Merrill, A. H. Jr; Hoel, M.; Wang, E.; Mullins, R. E.; Hargrove, J. L.; Jones, D. P.; Popova, I. A.; Merrill AH, J. r. (Principal Investigator)

1990-01-01

To determine the possible biochemical effects of prolonged weightlessness on liver function, samples of liver from rats that had flown aboard Cosmos 1887 were analyzed for protein, glycogen, and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. Among the parameters measured, the major differences were elevations in the glycogen content and hydroxymethylglutaryl-CoA (HMG-CoA) reductase activities for the rats flown on Cosmos 1887 and decreases in the amount of microsomal cytochrome P-450 and the activities of aniline hydroxylase and ethylmorphine N-demethylase, cytochrome P-450-dependent enzymes. These results support the earlier finding of differences in these parameters and suggest that altered hepatic function could be important during spaceflight and/or the postflight recovery period.

Management of pain in chronic pancreatitis with emphasis on exogenous pancreatic enzymes.

PubMed

Hobbs, Paul M; Johnson, William G; Graham, David Y

2016-08-06

One of the most challenging issues arising in patients with chronic pancreatitis is the management of abdominal pain. Many competing theories exist to explain pancreatic pain including ductal hypertension from strictures and stones, increased interstitial pressure from glandular fibrosis, pancreatic neuritis, and ischemia. This clinical problem is superimposed on a background of reduced enzyme secretion and altered feedback mechanisms. Throughout history, investigators have used these theories to devise methods to combat chronic pancreatic pain including: Lifestyle measures, antioxidants, analgesics, administration of exogenous pancreatic enzymes, endoscopic drainage procedures, and surgical drainage and resection procedures. While the value of each modality has been debated over the years, pancreatic enzyme therapy remains a viable option. Enzyme therapy restores active enzymes to the small bowel and targets the altered feedback mechanism that lead to increased pancreatic ductal and tissue pressures, ischemia, and pain. Here, we review the mechanisms and treatments for chronic pancreatic pain with a specific focus on pancreatic enzyme replacement therapy. We also discuss different approaches to overcoming a lack of clinical response update ideas for studies needed to improve the clinical use of pancreatic enzymes to ameliorate pancreatic pain.

Management of pain in chronic pancreatitis with emphasis on exogenous pancreatic enzymes

PubMed Central

Hobbs, Paul M; Johnson, William G; Graham, David Y

2016-01-01

One of the most challenging issues arising in patients with chronic pancreatitis is the management of abdominal pain. Many competing theories exist to explain pancreatic pain including ductal hypertension from strictures and stones, increased interstitial pressure from glandular fibrosis, pancreatic neuritis, and ischemia. This clinical problem is superimposed on a background of reduced enzyme secretion and altered feedback mechanisms. Throughout history, investigators have used these theories to devise methods to combat chronic pancreatic pain including: Lifestyle measures, antioxidants, analgesics, administration of exogenous pancreatic enzymes, endoscopic drainage procedures, and surgical drainage and resection procedures. While the value of each modality has been debated over the years, pancreatic enzyme therapy remains a viable option. Enzyme therapy restores active enzymes to the small bowel and targets the altered feedback mechanism that lead to increased pancreatic ductal and tissue pressures, ischemia, and pain. Here, we review the mechanisms and treatments for chronic pancreatic pain with a specific focus on pancreatic enzyme replacement therapy. We also discuss different approaches to overcoming a lack of clinical response update ideas for studies needed to improve the clinical use of pancreatic enzymes to ameliorate pancreatic pain. PMID:27602238

[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.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

PubMed

VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

2016-12-01

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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.

MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth.

PubMed

Delorme, Vincent; Diomandé, Sadia V; Dedieu, Luc; Cavalier, Jean-François; Carrière, Frédéric; Kremer, Laurent; Leclaire, Julien; Fotiadu, Frédéric; Canaan, Stéphane

2012-01-01

Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.

MmPPOX Inhibits Mycobacterium tuberculosis Lipolytic Enzymes Belonging to the Hormone-Sensitive Lipase Family and Alters Mycobacterial Growth

PubMed Central

Delorme, Vincent; Diomandé, Sadia V.; Dedieu, Luc; Cavalier, Jean-François; Carrière, Frédéric; Kremer, Laurent; Leclaire, Julien; Fotiadu, Frédéric; Canaan, Stéphane

2012-01-01

Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth. PMID:23029536

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis*

PubMed Central

Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya; Sankaran, Banumathi; Gilbert, Hiram F.; Prasad, B. V. Venkataram; Palzkill, Timothy

2015-01-01

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism. PMID:25713062

Monocyte Tumor Necrosis Factor-α–Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation*

PubMed Central

O’Callaghan, David J. P.; O’Dea, Kieran P.; Scott, Alasdair J.; Takata, Masao

2015-01-01

Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α–converting enzyme baseline and inducible activity profiles. Design: Observational clinical study. Setting: Mixed surgical/medical teaching hospital ICU. Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. Interventions: None. Measurements and Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α–converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients’ monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α–converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α–converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α–converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α–converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α–converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide. Conclusions: Monocyte tumor necrosis factor-α–converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α–converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis. PMID:25867908

Non-Alcoholic Fatty Liver Disease (NAFLD) - Pathogenesis, Classification, and Effect on Drug Metabolizing Enzymes and Transporters

PubMed Central

Cobbina, Enoch; Akhlaghi, Fatemeh

2017-01-01

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the presence of steatosis in more than 5 % of hepatocytes with little or no alcohol consumption. Insulin resistance, the metabolic syndrome or type 2 diabetes and genetic variants of PNPLA3 or TM6SF2 seem to play a role in the pathogenesis of NAFLD. The pathological progression of NAFLD follows tentatively a ‘three-hit’ process namely steatosis, lipotoxicity and inflammation. The presence of steatosis, oxidative stress and inflammatory mediators like TNF-α and IL-6 have been implicated in the alterations of nuclear factors such as CAR, PXR, PPAR-α in NAFLD. These factors may results in altered expression and activity of drug metabolizing enzymes (DMEs) or transporters. Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 are more consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas the activity of CYP2E1 and the efflux transporter MRP3 are up-regulated. However, it is not clear how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug variability in patients and could have serious implications for the safety and efficacy of xenobiotics. In this review, we summarize the effects of NAFLD on the regulation, expression and activity of major drug metabolizing enzymes and transporters. We also discuss the potential mechanisms underlying these alterations. PMID:28303724

The Role of Oxysterols in a Computational Steroidogenesis Model of Human H295R Cells to Improve Predictability of Biochemical Responses to Endocrine Disruptors

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine disruptors (...

Effect of starvation and refeeding on digestive enzyme activities in sturgeon (Acipenser naccarii) and trout (Oncorhynchus mykiss).

PubMed

Furné, Miriam; García-Gallego, Manuel; Hidalgo, M Carmen; Morales, Amalia E; Domezain, Alberto; Domezain, Julio; Sanz, Ana

2008-04-01

The digestive enzyme activities were determined in Adriatic sturgeon and rainbow trout during starvation and refeeding period. Overall, the digestive enzyme activities are affected in the same sense in both species. The protease and lipase activities were decreased later than amylase activity. Even after 1 month of starvation, both species would be prepared to digest protein and lipids in an effective way. After 72 days of starvation, the digestive machinery of the sturgeon and of the trout shows an altered capacity to digest macronutrients. The capacity to digest proteins and lipids, after 60 days of refeeding, begins to become re-established in sturgeon and trout. In contrast, in this period, the capacity to digest carbohydrates remains depressed in both species.

Chokeberry (Aronia melanocarpa) juice modulates 7,12-dimethylbenz[a]anthracene induced hepatic but not mammary gland phase I and II enzymes in female rats.

PubMed

Szaefer, Hanna; Krajka-Kuźniak, Violetta; Ignatowicz, Ewa; Adamska, Teresa; Baer-Dubowska, Wanda

2011-03-01

Chokeberry is a rich source of procyanidins known to have several types of biological activity including anticarcinogenic potential in experimental models. In this study we examined the effect of chokeberry juice on the hepatic and mammary gland carcinogen metabolizing enzyme expression altered by the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley rats were gavaged with chokeberry juice (8 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. Pretreatment with chokeberry juice reduced the activity of CYP1A1 and increased that of CYP2B involved in metabolic activation/detoxication of DMBA in rat liver, as well as expression and activity of phase II enzymes. Chokeberry juice had no effect on these parameters in the mammary gland and DMBA induced DNA damage in rat blood cells. These results together with our earlier observations indicate that metabolic alterations induced by chokeberry feeding are tissue specific and depend on the class of carcinogen. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of the Membrane-Bound Succinic Dehydrogenase of Micrococcus lysodeikticus

PubMed Central

Pollock, Jerry J.; Linder, Regina; Salton, Milton R. J.

1971-01-01

The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 × g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca2+ and Mg2+ exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents. Images PMID:4327510

Characterization of the membrane-bound succinic dehydrogenase of Micrococcus lysodeikticus.

PubMed

Pollock, J J; Linder, R; Salton, M R

1971-07-01

The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 x g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca(2+) and Mg(2+) exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents.

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.

Soil ecosystem functioning under climate change: plant species and community effects

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

Kardol, Paul; Cregger, Melissa; Campany, Courtney E

2010-01-01

Feedbacks of terrestrial ecosystems to climate change depend on soil ecosystem dynamics. Soil ecosystems can directly and indirectly respond to climate change. For example, warming directly alters microbial communities by increasing their activity. Climate change may also alter plant community composition, thus indirectly altering the microbial communities that feed on their inputs. To better understand how climate change may directly and indirectly alter soil ecosystem functioning, we investigated old-field plant community and soil ecosystem responses to single and combined effects of elevated [CO2], warming, and water availability. Specifically, we collected soils at the plot level (plant community soils), and beneathmore » dominant plant species (plant-specific soils). We used microbial enzyme activities and soil nematodes as indicators for soil ecosystem functioning. Our study resulted in two main findings: 1) Overall, while there were some interactions, water, relative to increases in [CO2] and warming, had the largest impact on plant community composition, soil enzyme activities, and soil nematodes. Multiple climate change factors can interact to shape ecosystems, but in this case, those interactions were largely driven by changes in water availability. 2) Indirect effects of climate change, via changes in plant communities, had a significant impact on soil ecosystem functioning and this impact was not obvious when looking at plant community soils. Climate change effects on enzyme activities and soil nematode abundance and community structure strongly differed between plant community soils and plant-specific soils, but also within plant-specific soils. In sum, these results indicate that accurate assessments of climate change impacts on soil ecosystem functioning require incorporating the concurrent changes in plant function and plant community composition. Climate change-induced shifts in plant community composition will likely modify or counteract the direct impact of climate change on soil ecosystem functioning, and hence, these indirect effects should be taken into account when predicting how climate change will alter ecosystem functioning.« less

Soil ecosystem functioning under climate change: plant species and community effects.

PubMed

Kardol, Paul; Cregger, Melissa A; Campany, Courtney E; Classen, Aimee T

2010-03-01

Feedbacks of terrestrial ecosystems to atmospheric and climate change depend on soil ecosystem dynamics. Soil ecosystems can directly and indirectly respond to climate change. For example, warming directly alters microbial communities by increasing their activity. Climate change may also alter plant community composition, thus indirectly altering the soil communities that depend on their inputs. To better understand how climate change may directly and indirectly alter soil ecosystem functioning, we investigated old-field plant community and soil ecosystem responses to single and combined effects of elevated [CO2], warming, and precipitation in Tennessee (USA). Specifically, we collected soils at the plot level (plant community soils) and beneath dominant plant species (plant-specific soils). We used microbial enzyme activities and soil nematodes as indicators for soil ecosystem functioning. Our study resulted in two main findings: (1) Overall, while there were some interactions, water, relative to increases in [CO2] and warming, had the largest impact on plant community composition, soil enzyme activity, and soil nematodes. Multiple climate-change factors can interact to shape ecosystems, but in our study, those interactions were largely driven by changes in water. (2) Indirect effects of climate change, via changes in plant communities, had a significant impact on soil ecosystem functioning, and this impact was not obvious when looking at plant community soils. Climate-change effects on enzyme activities and soil nematode abundance and community structure strongly differed between plant community soils and plant-specific soils, but also within plant-specific soils. These results indicate that accurate assessments of climate-change impacts on soil ecosystem functioning require incorporating the concurrent changes in plant function and plant community composition. Climate-change-induced shifts in plant community composition will likely modify or counteract the direct impact of atmospheric and climate change on soil ecosystem functioning, and hence, these indirect effects should be taken into account when predicting the manner in which global change will alter ecosystem functioning.

Ameliorating effect of Semecarpus anacardium Linn. nut milk extract on altered glucose metabolism in high fat diet STZ induced type 2 diabetic rats.

PubMed

Khan, Haseena Banu Hedayathullah; Vinayagam, Kaladevi Siddhi; Palanivelu, Shanthi; Panchanadham, Sachdanandam

2012-12-01

To explore the protective effect of the drug Semecarpus anacardium (S. anacardium)on altered glucose metabolism in diabetic rats. Type 2 diabetes mellitus was induced by feeding rats with high fat diet followed by single intraperitoneal injection of streptozotocin (STZ) (35 mg/kg b.w.). Seven days after STZ induction, diabetic rats received nut milk extract of S. anacardium Linn. nut milk extract orally at a dosage of 200 mg/kg daily for 4 weeks. The effect of nut milk extract of S. anacardium on blood glucose, plasma insulin, glucose metabolising enzymes and GSK were studied. Treatment with SA extract showed a significant reduction in blood glucose levels and increase in plasma insulin levels and also increase in HOMA - β and decrease in HOMA -IR. The drug significantly increased the activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase activity and increased the glycogen content in liver of diabetic rats while reducing the activities of gluconeogenic enzymes. The drug also effectively ameliorated the alterations in GSK-3 mRNA expression. Overall, the present study demonstrates the possible mechanism of glucose regulation of S. anacardium suggestive of its therapeutic potential for the management of diabetes mellitus. Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Altered sucrose synthase and invertase expression affects the local and systemic sugar metabolism of nematode-infected Arabidopsis thaliana plants.

PubMed

Cabello, Susana; Lorenz, Cindy; Crespo, Sara; Cabrera, Javier; Ludwig, Roland; Escobar, Carolina; Hofmann, Julia

2014-01-01

Sedentary endoparasitic nematodes of plants induce highly specific feeding cells in the root central cylinder. From these, the obligate parasites withdraw all required nutrients. The feeding cells were described as sink tissues in the plant's circulation system that are supplied with phloem-derived solutes such as sugars. Currently, there are several publications describing mechanisms of sugar import into the feeding cells. However, sugar processing has not been studied so far. Thus, in the present work, the roles of the sucrose-cleaving enzymes sucrose synthases (SUS) and invertases (INV) in the development of Heterodera schachtii were studied. Gene expression analyses indicate that both enzymes are regulated transcriptionally. Nematode development was enhanced on multiple INV and SUS mutants. Syncytia of these mutants were characterized by altered enzyme activity and changing sugar pool sizes. Further, the analyses revealed systemically affected sugar levels and enzyme activities in the shoots of the tested mutants, suggesting changes in the source-sink relationship. Finally, the development of the root-knot nematode Meloidogyne javanica was studied in different INV and SUS mutants and wild-type Arabidopsis plants. Similar effects on the development of both sedentary endoparasitic nematode species (root-knot and cyst nematode) were observed, suggesting a more general role of sucrose-degrading enzymes during plant-nematode interactions.

Nanoribbon field-effect transistors as direct and label-free sensors of enzyme-substrate interactions

NASA Astrophysics Data System (ADS)

Mu, Luye; Droujinine, Ilia; Rajan, Nitin; Sawtelle, Sonya; Reed, Mark

2015-03-01

The ability to measure enzyme-substrate interactions is essential in areas such as diagnostics, treatment, and biochemical screens. Many enzymatic reactions alter the pH of its environment, suggesting of a simple and direct method for detection. We show the ability of Al2O3-coated Si nanoribbon field-effect transistor biosensors to sensitively measure various aspects of enzyme-substrate interactions through measuring the pH. Urea in phosphate buffered saline (PBS) and penicillinase in PBS and urine were measured to limits of <200 μM and 0.02 units/mL, respectively. We also show the ability to extract accurate kinetics from the interaction of acetylcholine and its esterase. Prior work on FET sensors has been limited by the use of surface functionalization, which not only alters enzyme-substrate affinity, but also makes enzyme activity quantification difficult. Our method involves direct detection of reactions in solution without requiring alteration to the reactants, allowing us to obtain repeatable results and sensitive limits of detection. This method is a simple, inexpensive, and effective platform for detection of enzymatic reactions, and can be readily generalized to many unrelated classes of reactants. This work was supported in part by U.S. Army Research Office and Air Force Research Laboratory.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

PubMed Central

Kanade, Santosh R.; Paul, Beena; Rao, A. G. Appu; Gowda, Lalitha R.

2006-01-01

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

Alteration of the Expression of Pesticide-Metabolizing Enzymes in Pregnant Mice: Potential Role in the Increased Vulnerability of the Developing Brain

PubMed Central

Fortin, Marie C.; Aleksunes, Lauren M.

2013-01-01

Studies on therapeutic drug disposition in humans have shown significant alterations as the result of pregnancy. However, it is not known whether pesticide metabolic capacity changes throughout pregnancy, which could affect exposure of the developing brain. We sought to determine the effect of pregnancy on the expression of hepatic enzymes involved in the metabolism of pesticides. Livers were collected from virgin and pregnant C57BL/6 mice at gestational days (GD)7, GD11, GD14, GD17, and postpartum days (PD)1, PD15, and PD30. Relative mRNA expression of several enzymes involved in the metabolism of pesticides, including hepatic cytochromes (Cyp) P450s, carboxylesterases (Ces), and paraoxonase 1 (Pon1), were assessed in mice during gestation and the postpartum period. Compared with virgin mice, alterations in the expression occurred at multiple time points, with the largest changes observed on GD14. At this time point, the expression of most of the Cyps involved in pesticide metabolism in the liver (Cyp1a2, Cyp2d22, Cyp2c37, Cyp2c50, Cyp2c54, and Cyp3a11) were downregulated by 30% or more. Expression of various Ces isoforms and Pon1 were also decreased along with Pon1 activity. These data demonstrate significant alterations in the expression of key enzymes that detoxify pesticides during pregnancy, which could alter exposure of developing animals to these chemicals. PMID:23223497

Radiation-induced enzyme efflux from rat heart: sedentary animals. [Gamma radiation, lactate dehydrogenase, creative kinase, glutamate oxaloacetate transaminase

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

MacWilliam, L.D.; Bhakthan, N.M.G.

1976-01-01

Serum levels of lactate dehydrogenase, creatine kinase, and glutamate oxaloacetate transaminase show initial elevations within 12 hr of exposure to 2,000 rads of ..gamma..-radiation to the thoracic region of rats. Significant decreases in heart muscle homogenate levels of these enzymes parallel initial elevations in the serum and may suggest that enhanced leakage of enzymes is a consequence of radiation injury to heart muscle. Insignificant alterations in mitochondrial glutamate oxaloacetate transaminase levels after exposure indicate that in vivo injury to the mitochondria from therapeutic levels of ..gamma..-radiation is questionable. The results support the contention that ionizing radiation instigates alterations in themore » dynamic permeability of membranes, allowing leakage of biologically active material out of the injured cell.« less

Chronic inhibition of Ca(2+)/calmodulin kinase II activity in the pilocarpine model of epilepsy.

PubMed

Churn, S B; Kochan, L D; DeLorenzo, R J

2000-09-01

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.

Growth at elevated ozone or elevated carbon dioxide concentration alters antioxidant capacity and response to acute oxidative stress in soybean (Glycine max)

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

Gillespie, K.M.; Rogers, A.; Ainsworth, E. A.

2011-01-31

Soybeans (Glycine max Merr.) were grown at elevated carbon dioxide concentration ([CO{sub 2}]) or chronic elevated ozone concentration ([O{sub 3}]; 90 ppb), and then exposed to an acute O{sub 3} stress (200 ppb for 4 h) in order to test the hypothesis that the atmospheric environment alters the total antioxidant capacity of plants, and their capacity to respond to an acute oxidative stress. Total antioxidant metabolism, antioxidant enzyme activity, and antioxidant transcript abundance were characterized before, immediately after, and during recovery from the acute O{sub 3} treatment. Growth at chronic elevated [O{sub 3}] increased the total antioxidant capacity of plants,more » while growth at elevated [CO{sub 2}] decreased the total antioxidant capacity. Changes in total antioxidant capacity were matched by changes in ascorbate content, but not phenolic content. The growth environment significantly altered the pattern of antioxidant transcript and enzyme response to the acute O{sub 3} stress. Following the acute oxidative stress, there was an immediate transcriptional reprogramming that allowed for maintained or increased antioxidant enzyme activities in plants grown at elevated [O{sub 3}]. Growth at elevated [CO{sub 2}] appeared to increase the response of antioxidant enzymes to acute oxidative stress, but dampened and delayed the transcriptional response. These results provide evidence that the growth environment alters the antioxidant system, the immediate response to an acute oxidative stress, and the timing over which plants return to initial antioxidant levels. The results also indicate that future elevated [CO{sub 2}] and [O{sub 3}] will differentially affect the antioxidant system.« less

Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

PubMed

Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

2015-04-01

The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

Bendiocarb induced histopathological and biochemical alterations in rat liver and preventive role of vitamins C and E.

PubMed

Apaydin, Fatma Gökçe; Baş, Hatice; Kalender, Suna; Kalender, Yusuf

2017-01-01

In this study, biochemical changes and histological structure of rat liver after bendiocarb administration and possible preventive effects of vitamins C and E were studied. The animals were given with bendiocarb, vitamin C and vitamin E, daily 0,8mg/kg of body weight (bw), 100mg/kg-bw and 100mg/kg-bw for 28days, respectively. Lipid peroxidation, antioxidant enzyme activities, histological alterations and antioxidant capacity assays of liver and also liver function tests and lipid profile were measured. Bendiocarb treatment decreased the antioxidant enzyme activities, FRAP and TEAC values and increased malondialdehyde levels compared to control. Also, there were statistically significant alterations in liver function tests, lipid profile parameters and histopathological changes in bendiocarb treated groups. Vitamins C and E showed protective effects against examining parameters. According to results we can say that co-treatment of vitamin C and vitamin E may be more effective than use of them alone. Copyright © 2016 Elsevier B.V. All rights reserved.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

PubMed Central

Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

2015-01-01

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

Transcriptional switches in the control of macronutrient metabolism.

PubMed

Wise, Alan

2008-06-01

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

Mitochondrial oxidative enzyme activity in individual fibre types in hypo- and hyperthyroid rat skeletal muscles.

PubMed

Johnson, M A; Turnbull, D M

1984-04-01

Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa.

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.

Ameliorative Influence of Green Tea Extract on Copper Nanoparticle-Induced Hepatotoxicity in Rats

NASA Astrophysics Data System (ADS)

Ibrahim, Marwa A.; Khalaf, A. A.; Galal, Mona K.; Ogaly, Hanan A.; H. M. Hassan, Azza

2015-09-01

The potential toxicity of copper nanoparticles (CNPs) to the human health and environment remains a critical issue. In the present study, we investigated the protective influence of an aqueous extract of green tea leaves (GTE) against CNPs-induced (20-30 nm) hepatotoxicity. Four different groups of rats were used: group I was the control, group II received CNPs (40 mg/kg BW), group III received CNPs plus GTE, and group IV received GTE alone. We highlighted the hepatoprotective effect of GTE against CNPs toxicity through monitoring the alteration of liver enzyme activity, antioxidant defense mechanism, histopathological alterations, and DNA damage evaluation. The rats that were given CNPs only had a highly significant elevation in liver enzymes, alteration in oxidant-antioxidant balance, and severe pathological changes. In addition, we detected a significant elevation of DNA fragmentation percentage, marked DNA laddering, and significance over expression of both caspase-3 and Bax proteins. The findings for group III clarify the efficacy of GTE as a hepatoprotectant on CNPs through improving the liver enzyme activity, antioxidant status, as well as suppressing DNA fragmentation and the expression of the caspase-3 and Bax proteins. In conclusion, GTE was proved to be a potential hepatoprotective additive as it significantly ameliorates the hepatotoxicity and apoptosis induced by CNPs.

Upregulation of Glycolytic Enzymes, Mitochondrial Dysfunction and Increased Cytotoxicity in Glial Cells Treated with Alzheimer’s Disease Plasma

PubMed Central

Jayasena, Tharusha; Poljak, Anne; Braidy, Nady; Smythe, George; Raftery, Mark; Hill, Mark; Brodaty, Henry; Trollor, Julian; Kochan, Nicole; Sachdev, Perminder

2015-01-01

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia. PMID:25785936

Global changes alter soil fungal communities and alter rates of organic matter decomposition

NASA Astrophysics Data System (ADS)

Moore, J.; Frey, S. D.

2016-12-01

Global changes - such as warming, more frequent and severe droughts, increasing atmospheric CO2, and increasing nitrogen (N) deposition rates - are altering ecosystem processes. The balance between soil carbon (C) accumulation and decomposition is determined in large part by the activity and biomass of detrital organisms, namely soil fungi, and yet their sensitivity to global changes remains unresolved. We present results from a meta-analysis of 200+ studies spanning manipulative and observational field experiments to quantify fungal responses to global change and expected consequences for ecosystem C dynamics. Warming altered the functional soil microbial community by reducing the ratio of fungi to bacteria (f:b) total fungal biomass. Additionally, warming reduced lignolytic enzyme activity generally by one-third. Simulated N deposition affected f:b differently than warming, but the effect on fungal biomass and activity was similar. The effect of N-enrichment on f:b was contingent upon ecosystem type; f:b increased in alpine meadows and heathlands but decreased in temperate forests following N-enrichment. Across ecosystems, fungal biomass marginally declined by 8% in N-enriched soils. In general, N-enrichment reduced fungal lignolytic enzyme activity, which could explain why soil C accumulates in some ecosystems following warming and N-enrichment. Several global change experiments have reported the surprising result that soil C builds up following increases in temperature and N deposition rates. While site-specific studies have examined the role of soil fungi in ecosystem responses to global change, we present the first meta-analysis documenting general patterns of global change impacts on soil fungal communities, biomass, and activity. In sum, we provide evidence that soil microbial community shifts and activity plays a large part in ecosystem responses to global changes, and have the potential to alter the magnitude of the C-climate feedback.

Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

PubMed

Zera, Anthony J; Zhao, Zhangwu

2003-03-01

Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Soybean cotyledon starch metabolism is sensitive to altered gravity conditions

NASA Technical Reports Server (NTRS)

Brown, C. S.; Piastuch, W. C.; Knott, W. M.

1994-01-01

We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.

Piper species protect cardiac, hepatic and renal antioxidant status of atherogenic diet fed hamsters.

PubMed

Agbor, Gabriel A; Akinfiresoye, Luli; Sortino, Julianne; Johnson, Robert; Vinson, Joe A

2012-10-01

Pre-clinical and clinical studies points to the use of antioxidants as an effective measure to reduce the progression of oxidative stress related disorders. The present study evaluate the effect of three Piper species (Piper guineense, Piper nigrum and Piper umbellatum) for the protection of cardiac, hepatic and renal antioxidant status of atherogenic diet fed hamsters. Hamsters were classified into eight groups: a normal control, atherogenic control and six other experimental groups (fed atherogenic diet supplemented with different doses of P. nigrum, P. guineense and P. umbellatum (1 and 0.25 g/kg) for 12 weeks. At the end of the feeding period the heart, liver and kidney from each group were analyzed for lipid profile and antioxidant enzymes activities. Atherogenic diet induced a significant (P<0.001) increase in the lipid profile across the board and equally significantly altered the antioxidant enzyme activities. Supplementation with Piper species significantly inhibited the alteration effect of atherogenic diet on the lipid profile and antioxidant enzymes activities. The Piper extracts may possess an antioxidant protective role against atherogenic diet induced oxidative stress in cardiac, hepatic and renal tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

EPA Science Inventory

Abstract

Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

NASA Astrophysics Data System (ADS)

Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

2014-05-01

Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

Interaction of firefly luciferase and silver nanoparticles and its impact on enzyme activity

NASA Astrophysics Data System (ADS)

Käkinen, Aleksandr; Ding, Feng; Chen, Pengyu; Mortimer, Monika; Kahru, Anne; Ke, Pu Chun

2013-08-01

We report on the dose-dependent inhibition of firefly luciferase activity induced by exposure of the enzyme to 20 nm citrate-coated silver nanoparticles (AgNPs). The inhibition mechanism was examined by characterizing the physicochemical properties and biophysical interactions of the enzyme and the AgNPs. Consistently, binding of the enzyme induced an increase in zeta potential from -22 to 6 mV for the AgNPs, triggered a red-shift of 44 nm in the absorbance peak of the AgNPs, and rendered a ‘protein corona’ of 20 nm in thickness on the nanoparticle surfaces. However, the secondary structures of the enzyme were only marginally affected upon formation of the protein corona, as verified by circular dichroism spectroscopy measurement and multiscale discrete molecular dynamics simulations. Rather, inductively coupled plasma mass spectrometry measurement revealed a significant ion release from the AgNPs. The released silver ions could readily react with the cysteine residues and N-groups of the enzyme to alter the physicochemical environment of their neighboring catalytic site and subsequently impair the enzymatic activity.

Alterations of the levels of primary antioxidant enzymes in different grades of human astrocytoma tissues.

PubMed

Yen, Hsiu-Chuan; Lin, Chih-Lung; Chen, Bing-Shian; Chen, Chih-Wei; Wei, Kuo-Chen; Yang, Mei-Lin; Hsu, Jee-Ching; Hsu, Yung-Hsing

2018-06-03

Malignant astrocytoma is the most commonly occurring brain tumor in humans. Oxidative stress is implicated in the development of cancers. Superoxide dismutase 2 (SOD2) was found to exert tumor suppressive effect in basic research, but increased SOD2 protein level was associated with higher aggressiveness of human astrocytomas. However, studies reporting alterations of antioxidant enzymes in human astrocytomas often employed less accurate methods or included different types of tumors. Here we analyzed the mRNA levels, activities, and protein levels of primary antioxidant enzymes in control brain tissues and various grades of astrocytomas obtained from 40 patients. SOD1 expression, SOD1 activity, and SOD1 protein level were lower in Grade IV astrocytomas. SOD2 expression was lower in low-grade (Grades I and II) and Grade III astrocytomas than in controls, but SOD2 expression and SOD2 protein level were higher in Grade IV astrocytomas than in Grade III astrocytomas. Although there was no change in SOD2 activity and a lower activity of citrate synthase (CS), the MnSOD:CS ratio increased in Grade IV astrocytomas compared with controls and low-grade astrocytomas. Furthermore, SOD1 activity, CS activity, SOD1 expression, GPX4 expression, and GPX4 protein level were inversely correlated with the malignancy, whereas catalase activity, catalase protein, SOD2 protein level, and the SOD2:CS ratio were positively correlated with the degree of malignancy. Lower SOD2:CS ratio was associated with poor outcomes for Grade IV astrocytomas. This is the first study to quantify changes of various primary antioxidant enzymes in different grades of astrocytomas at different levels concurrently in human astrocytomas.

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

PubMed

Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

2017-09-01

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

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

Nault, Rance, E-mail: naultran@msu.edu; Abdul-Fattah, Hiba; Mironov, Gleb G.

2013-08-15

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesismore » rates, Na{sup +}/K{sup +}-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na{sup +}/K{sup +}-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism.« less

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

PubMed

Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

2016-01-01

S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

PubMed

Suplatov, D A; Arzhanik, V K; Svedas, V K

2011-01-01

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

Selection for growth on 3-nitrotoluene by 2-nitrotoluene-utilizing Acidovorax sp. strain JS42 identifies nitroarene dioxygenases with altered specificities.

PubMed

Mahan, Kristina M; Penrod, Joseph T; Ju, Kou-San; Al Kass, Natascia; Tan, Watumesa A; Truong, Richard; Parales, Juanito V; Parales, Rebecca E

2015-01-01

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

PubMed

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Altered sucrose synthase and invertase expression affects the local and systemic sugar metabolism of nematode-infected Arabidopsis thaliana plants

PubMed Central

Hofmann, Julia

2014-01-01

Sedentary endoparasitic nematodes of plants induce highly specific feeding cells in the root central cylinder. From these, the obligate parasites withdraw all required nutrients. The feeding cells were described as sink tissues in the plant’s circulation system that are supplied with phloem-derived solutes such as sugars. Currently, there are several publications describing mechanisms of sugar import into the feeding cells. However, sugar processing has not been studied so far. Thus, in the present work, the roles of the sucrose-cleaving enzymes sucrose synthases (SUS) and invertases (INV) in the development of Heterodera schachtii were studied. Gene expression analyses indicate that both enzymes are regulated transcriptionally. Nematode development was enhanced on multiple INV and SUS mutants. Syncytia of these mutants were characterized by altered enzyme activity and changing sugar pool sizes. Further, the analyses revealed systemically affected sugar levels and enzyme activities in the shoots of the tested mutants, suggesting changes in the source–sink relationship. Finally, the development of the root-knot nematode Meloidogyne javanica was studied in different INV and SUS mutants and wild-type Arabidopsis plants. Similar effects on the development of both sedentary endoparasitic nematode species (root-knot and cyst nematode) were observed, suggesting a more general role of sucrose-degrading enzymes during plant–nematode interactions. PMID:24187419

Frequent alteration of the protein synthesis of enzymes for glucose metabolism in hepatocellular carcinomas.

PubMed

Shimizu, Takayuki; Inoue, Ken-ichi; Hachiya, Hiroyuki; Shibuya, Norisuke; Shimoda, Mitsugi; Kubota, Keiichi

2014-09-01

Cancer cells show enhanced glycolysis and inhibition of oxidative phosphorylation, even in the presence of sufficient oxygen (aerobic glycolysis). Glycolysis is much less efficient for energy production than oxidative phosphorylation, and the reason why cancer cells selectively use glycolysis remains unclear. Biospecimens were collected from 45 hepatocellular carcinoma patients. Protein samples were prepared through subcellular localization or whole-cell lysis. Protein synthesis was measured by SDS-PAGE and immunoblotting. mRNA transcription was measured using quantitative RT-PCR. Statistical correlation among immunoblotting data and clinicolaboratory factors were analyzed using SPSS. Enzymes for oxidative phosphorylation (SDHA and SDHB) were frequently decreased (56 and 48 % of patients, respectively) in hepatocellular carcinomas. The lowered amount of the SDH protein complex was rarely accompanied by stabilization of HIF1α and subsequent activation of the hypoxia response. On the other hand, protein synthesis of G6PD and TKT, enzymes critical for pentose phosphate pathway (PPP), was increased (in 45 and 55 % of patients, respectively), while that of ALDOA, an enzyme for mainstream glycolysis, was eliminated (in 55 % of patients). Alteration of protein synthesis was correlated with gene expression for G6PD and TKT, but not for TKTL1, ALDOA, SDHA or SDHB. Augmented transcription and synthesis of PPP enzymes were accompanied by nuclear accumulation of NRF2. Hepatocellular carcinomas divert glucose metabolism to the anabolic shunt by activating transcription factor NRF2.

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.

[Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans].

PubMed

Liber, E E; Dorozhko, A I; Pomortseva, N V

1978-06-01

The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.

Controlling enzymatic activity by immobilization on graphene oxide

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Halophiles and their enzymes: negativity put to good use.

PubMed

DasSarma, Shiladitya; DasSarma, Priya

2015-06-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

Halophiles and their enzymes: Negativity put to good use

PubMed Central

DasSarma, Shiladitya; DasSarma, Priya

2015-01-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. On-going efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. PMID:26066288

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

Alterations in the endocannabinoid system in the rat valproic acid model of autism.

PubMed

Kerr, D M; Downey, L; Conboy, M; Finn, D P; Roche, M

2013-07-15

The endocannabinoid system plays a crucial role in regulating emotionality and social behaviour, however it is unknown whether this system plays a role in symptoms associated with autism spectrum disorders. The current study evaluated if alterations in the endocannabinoid system accompany behavioural changes in the valproic acid (VPA) rat model of autism. Adolescent rats prenatally exposed to VPA exhibited impaired social investigatory behaviour, hypoalgesia and reduced lococmotor activity on exposure to a novel aversive arena. Levels of the endocananbinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG) in the hippocampus, frontal cortex or cerebellum were not altered in VPA- versus saline-exposed animals. However, the expression of mRNA for diacylglycerol lipase α, the enzyme primarily responsible for the synthesis of 2-AG, was reduced in the cerebellum of VPA-exposed rats. Furthermore, while the expression of mRNA for the 2-AG-catabolising enzyme monoacylglycerol lipase was reduced, the activity of this enzyme was increased, in the hippocampus of VPA-exposed animals. CB1 or CB2 receptor expression was not altered in any of the regions examined, however VPA-exposed rats exhibited reduced PPARα and GPR55 expression in the frontal cortex and PPARγ and GPR55 expression in the hippocampus, additional receptor targets of the endocannabinoids. Furthermore, tissue levels of the fatty acid amide hydrolase substrates, AEA, oleoylethanolamide and palmitoylethanolamide, were higher in the hippocampus of VPA-exposed rats immediately following social exposure. These data indicate that prenatal VPA exposure is associated with alterations in the brain's endocannabinoid system and support the hypothesis that endocannabinoid dysfunction may underlie behavioural abnormalities observed in autism spectrum disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

PubMed

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Chlorogenic acid ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes.

PubMed

Akila, Palaniyandi; Asaikumar, Lourthurani; Vennila, Lakshmanan

2017-01-01

This study was deliberated to aspire the effects of chlorogenic acid (CGA) against myocardial infarction (MI) induced by Isoproterenol (ISO), in a rat model. In the pathology of MI, enzymes released due to the mitochondrial and lysosomal lipid peroxidation play an integral role. Induction of rats with ISO (85mg/kg BW) for 2 consecutive days resulted in a significant decrease in the activities of heart mitochondrial enzymes isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). The activities of lysosomal enzymes (β- glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, cathepsin-B and cathepsin-D) were increased significantly in the heart tissue. A prominent expression of LDH 1 and LDH 2 isoenzymes in the serum were observed and changes in the Electrocardiographic (ECG) patterns were also recorded in the ISO-induced rats. The prior administrations of CGA (40mg/kg BW) for 19days markedly ameliorated ISO induced alterations in ECG and significantly restored the activities of all the above enzymes in the heart of ISO-induced rats, which substantiates the stress stabilizing action of CGA. Oral administration of CGA (40mg/kg BW) to normal rats did not show any significant changes. These biochemical functional alterations were supported by the histology of heart (Massion's trichrome and Picrosirius red staining for collagen formation). Thereupon, this study shows that 40mg/kg BW of CGA gives protection against ISO-induced MI and demonstrates that CGA has a significant effect in the protection of heart. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

PubMed Central

Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

2016-01-01

‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’. PMID:27385485

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.

Alterations in Krebs cycle enzyme activities and carbohydrate catabolism in two strains of Trypanosoma brucei during in vitro differentiation of their bloodstream to procyclic stages.

PubMed

Durieux, P O; Schütz, P; Brun, R; Köhler, P

1991-03-01

A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of solar irradiation on extracellular enzymes of Aeromonas proteolytica

NASA Technical Reports Server (NTRS)

Foster, B. G.

1973-01-01

The bacterium Aeromonas proteolytica was selected for studying the effects of solar irradiation on extracellular enzymes because it produces an endopeptidase that is capable of degrading proteins and a hemolysin that is active in lysing human erythrocytes. Possible alterations in the rate of enzyme production in response to the test conditions are currently underway and are not available for this preliminary report. Completed viability studies are indicative that little difference exists among the survival curves derived for cells exposed to various components of ultraviolet irradiation in space.

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans.

PubMed

Sarre, Aili; Ökvist, Mats; Klar, Tobias; Hall, David R; Smalås, Arne O; McSweeney, Sean; Timmins, Joanna; Moe, Elin

2015-08-01

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Use of Pressure Activation in Food Quality Improvement.

PubMed

Shigematsu, Toru

2015-01-01

Beside intensive studies on inactivation microorganisms by high hydrostatic pressure (HP) for food storage, pressure effects on property of food materials have also been studied based on knowledge in pressure effect on biomolecules. Pressure effects on biological membranes and mass transfer in cellular biological materials and on enzyme activity would give an idea that HP treatment can introduce two types of activations into food materials: improved mass transfer and enzyme activity. Studies focusing on these pressure activations on food materials were then reviewed. Rice flour with an exclusively fine mean particle size and small starch damage was obtained due to improved water absorption properties and/or enzyme activity by HP. HP treatment increased of free amino acids and γ-aminobutyric acid (GABA) in rice and soybeans due to improved proteolysis and amino acid metabolism. Improvement of antioxidant activity and alteration of polyphenolic-compounds composition in food materials were also demonstrated by HP treatment. The HP-induced activations on food materials could contribute towards processing technologies for food quality improvement.

Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes.

PubMed

McClelland, G B; Dalziel, A C; Fragoso, N M; Moyes, C D

2005-02-01

We investigated if seasonal changes in rainbow trout muscle energetics arise in response to seasonal changes in erythrocyte properties. We assessed if skeletal muscle mitochondrial enzymes changed (1) acutely in response to changes in erythrocyte abundance, or (2) seasonally when we altered the age profile of erythrocytes. Rainbow trout were treated with pheynylhydrazine, causing a 75% reduction in hematocrit within 4 days. After erythropoiesis had returned hematocrit to normal, treated and control fish were subjected to a seasonal cold acclimation regime to assess the impact of erythrocyte age on skeletal muscle remodeling. Anemia (i.e. phenylhydrazine treatment) did not alter the specific activities (U g(-1) tissue) of mitochondrial enzymes in white or red muscle. Anemic pretreatment did not alter the normal pattern of cold-induced mitochondrial proliferation in skeletal muscle, suggesting erythrocyte age was not an important influence on seasonal remodeling of muscle. Anemia and cold acclimation both induced a 25-30% increase in relative ventricular mass. The increase in relative ventricular mass with phenylhydrazine treatment was accompanied by a 35% increase in DNA content (mg DNA per ventricle), suggesting an increase in number of cells. In contrast, the increase in ventricular mass with cold temperature acclimation occurred without a change in DNA content (mg DNA per ventricle), suggesting an increase in cell size. Despite the major increases in relative ventricular mass, neither anemia nor seasonal acclimation had a major influence on the specific activities of a suite of mitochondrial enzymes in heart. Collectively, these studies argue against a role for erythrocyte dynamics in seasonal adaptive remodeling of skeletal muscle energetics.

Pharmacologic modulation of ACE2 expression.

PubMed

Soler, María José; Barrios, Clara; Oliva, Raymond; Batlle, Daniel

2008-10-01

Angiotensin-converting enzyme 2 (ACE2) is an enzymatically active homologue of angiotensin-converting enzyme that degrades angiotensin I, angiotensin II, and other peptides. Recent studies have shown that under pathologic conditions, ACE2 expression in the kidney is altered. In this review, we briefly summarize recent studies dealing with pharmacologic interventions that modulate ACE2 expression. ACE2 amplification may have a potential therapeutic role for kidney disease and hypertension.

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

PubMed Central

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Understanding and Improving the Activity of Flavin Dependent Halogenases via Random and Targeted Mutagenesis

PubMed Central

Andorfer, Mary C.

2018-01-01

Flavin dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes has been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications. PMID:29589959

A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells

PubMed Central

Bagley, Pamela J.; Selhub, Jacob

1998-01-01

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates. PMID:9789068

Copper Regulates Cyclic AMP-Dependent Lipolysis

PubMed Central

Krishnamoorthy, Lakshmi; Cotruvo, Joseph A.; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S.; Jia, Shang; Aron, Allegra T.; Ackerman, Cheri M.; Vander Wal, Mark N.; Guan, Timothy; Smaga, Lukas P.; Farhi, Samouil L.; New, Elizabeth J.; Lutsenko, Svetlana; Chang, Christopher J.

2016-01-01

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium, and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining the body's weight and energy stores. Utilizing a murine model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we demonstrate that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue within a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype. PMID:27272565

Assessment of biological effects of environmental pollution in Mersin Bay (Turkey, northeastern Mediterranean Sea) using Mullus barbatus and Liza ramada as target organisms.

PubMed

Yılmaz, Doruk; Kalay, Mustafa; Dönmez, Erdem; Yılmaz, Nejat

2016-01-01

The increasing emphasis on the assessment and monitoring of marine ecosystems has revealed the need to use appropriate biological indicators for these areas. Enzyme activities and histopathology are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of pollutant exposure. As part of an ecotoxicological assessment of Mersin Bay, EROD enzyme activity and histopathological response in selected organs and tissues of two species of fish, Mullus barbatus (red mullet) and Liza ramada (thinlip grey mullet), captured from area were examined. Pollutant (Organochlorines (OC), alkylphenols (APs) and BPA) levels and biomarker responses in tissue samples were evaluated together for their potential to alter the metabolism and cellular aspects in liver and gonad. Elevated induction of EROD activity and histopathological alterations in contaminated samples from Mersin Bay was observed compared to reference site indicating the exposure to potential pollutants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

USGS Publications Warehouse

Buhler, Donald R.; Benville, P.

1969-01-01

The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

Mammalian monoamine-oxidizing enzymes, with special reference to benzylamine oxidase in human tissues.

PubMed

Lewinsohn, R

1984-01-01

A review is presented of the monoamine-oxidizing enzymes with special reference to the activity of benzylamine oxidase (BzAO) in human tissues. Methods of study of amine oxidases, properties (chiefly of BzAO) and some problems concerning substrate and inhibitor specificity and multiple forms of monoamine oxidase (MAO) are surveyed. The substrate specificity of human plasma BzAO is compared with that of amine-oxidizing enzymes in plasma or serum of other species. Correlations of plasma BzAO and platelet MAO activity with clinical findings are discussed. The distribution of amine oxidase activities in solid human tissues is reviewed, in particular BzAO in blood vessels and richly-vascularized tissues, as well as kinetic constants and altered patterns of activity of BzAO in human atherosclerosis. Activities of the amine oxidases in non-vascular smooth muscle, in cultured cells, and in various tissues related to human gestation, are discussed. The present knowledge of BzAO is discussed in terms of its possible clinical relevance to several human disease states, and the importance of the enzyme in the human body.

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

PubMed

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

2012-07-01

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

Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

PubMed

Leurs, Melanie; Tiller, Joerg C

2017-01-01

The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

NASA Technical Reports Server (NTRS)

Ordoukhanian, Phillip; Joyce, Gerald F.

2002-01-01

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Modulation of Metabolic Detoxification Pathways Using Foods and Food-Derived Components: A Scientific Review with Clinical Application.

PubMed

Hodges, Romilly E; Minich, Deanna M

2015-01-01

Research into human biotransformation and elimination systems continues to evolve. Various clinical and in vivo studies have been undertaken to evaluate the effects of foods and food-derived components on the activity of detoxification pathways, including phase I cytochrome P450 enzymes, phase II conjugation enzymes, Nrf2 signaling, and metallothionein. This review summarizes the research in this area to date, highlighting the potential for foods and nutrients to support and/or modulate detoxification functions. Clinical applications to alter detoxification pathway activity and improve patient outcomes are considered, drawing on the growing understanding of the relationship between detoxification functions and different disease states, genetic polymorphisms, and drug-nutrient interactions. Some caution is recommended, however, due to the limitations of current research as well as indications that many nutrients exert biphasic, dose-dependent effects and that genetic polymorphisms may alter outcomes. A whole-foods approach may, therefore, be prudent.

Modulation of Metabolic Detoxification Pathways Using Foods and Food-Derived Components: A Scientific Review with Clinical Application

PubMed Central

Hodges, Romilly E.; Minich, Deanna M.

2015-01-01

Research into human biotransformation and elimination systems continues to evolve. Various clinical and in vivo studies have been undertaken to evaluate the effects of foods and food-derived components on the activity of detoxification pathways, including phase I cytochrome P450 enzymes, phase II conjugation enzymes, Nrf2 signaling, and metallothionein. This review summarizes the research in this area to date, highlighting the potential for foods and nutrients to support and/or modulate detoxification functions. Clinical applications to alter detoxification pathway activity and improve patient outcomes are considered, drawing on the growing understanding of the relationship between detoxification functions and different disease states, genetic polymorphisms, and drug-nutrient interactions. Some caution is recommended, however, due to the limitations of current research as well as indications that many nutrients exert biphasic, dose-dependent effects and that genetic polymorphisms may alter outcomes. A whole-foods approach may, therefore, be prudent. PMID:26167297

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

PubMed Central

Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

2012-01-01

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

EMF radiations (1800 MHz)-inhibited early seedling growth of maize (Zea mays) involves alterations in starch and sucrose metabolism.

PubMed

Kumar, Arvind; Singh, Harminder Pal; Batish, Daizy R; Kaur, Shalinder; Kohli, Ravinder Kumar

2016-07-01

The present study investigated the impact of 1800-MHz electromagnetic field radiations (EMF-r), widely used in mobile communication, on the growth and activity of starch-, sucrose-, and phosphate-hydrolyzing enzymes in Zea mays seedlings. We exposed Z. mays to modulated continuous wave homogenous EMF-r at specific absorption rate (SAR) of 1.69±0.0 × 10(-1) W kg(-1) for ½, 1, 2, and 4 h. The analysis of seedlings after 7 days revealed that short-term exposure did not induce any significant change, while longer exposure of 4 h caused significant growth and biochemical alterations. There was a reduction in the root and coleoptile length with more pronounced effect on coleoptile growth (23 % reduction on 4-h exposure). The contents of photosynthetic pigments and total carbohydrates declined by 13 and 18 %, respectively, in 4-h exposure treatments compared to unexposed control. The activity of starch-hydrolyzing enzymes-α- and β-amylases-increased by ∼92 and 94 %, respectively, at an exposure duration of 4 h, over that in the control. In response to 4-h exposure treatment, the activity of sucrolytic enzymes-acid invertases and alkaline invertases-was increased by 88 and 266 %, whereas the specific activities of phosphohydrolytic enzymes (acid phosphatases and alkaline phosphatases) showed initial increase up to ≤2 h duration and then declined at >2 h exposure duration. The study concludes that EMF-r-inhibited seedling growth of Z. mays involves interference with starch and sucrose metabolism.

Human T-lymphotropic virus proteins and post-translational modification pathways

PubMed Central

Bidoia, Carlo

2012-01-01

Cell life from the cell cycle to the signaling transduction and response to stimuli is finely tuned by protein post-translational modifications (PTMs). PTMs alter the conformation, the stability, the localization, and hence the pattern of interactions of the targeted protein. Cell pathways involve the activation of enzymes, like kinases, ligases and transferases, that, once activated, act on many proteins simultaneously, altering the state of the cell and triggering the processes they are involved in. Viruses enter a balanced system and hijack the cell, exploiting the potential of PTMs either to activate viral encoded proteins or to alter cellular pathways, with the ultimate consequence to perpetuate through their replication. Human T-lymphotropic virus type 1 (HTLV-1) is known to be highly oncogenic and associates with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis and other inflammatory pathological conditions. HTLV-1 protein activity is controlled by PTMs and, in turn, viral activity is associated with the modulation of cellular pathways based on PTMs. More knowledge is acquired about the PTMs involved in the activation of its proteins, like Tax, Rex, p12, p13, p30, HTLV-I basic leucine zipper factor and Gag. However, more has to be understood at the biochemical level in order to counteract the associated fatal outcomes. This review will focus on known PTMs that directly modify HTLV-1 components and on enzymes whose activity is modulated by viral proteins. PMID:24175216

[Status of the lipid peroxidation system in the tissues of rats following a 7-day flight on the Kosmos-1667 biosatellite].

PubMed

Delenian, N V; Markin, A A

1989-01-01

Rats flown for 7 days on Cosmos-1667 were for the first time used to measure antioxidative enzymes (superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase), lipid peroxidation products (diene conjugates, malonic dialdehyde, Schiff bases) and tocopherol. Enhanced lipid peroxidation in the heart was completely compensated by activation of antioxidative enzymes. The content of all lipid peroxidation products measured in the liver increased; this was accompanied by a decrease of glutathione peroxidase and an increase of superoxide dismutase activities. It is suggested that lipid peroxidation was activated in response to altered gravity.

Role of nitric oxide and antioxidant enzymes in the pathogenesis of oral cancer.

PubMed

Patel, Jayendrakumar B; Shah, Franky D; Shukla, Shilin N; Shah, Pankaj M; Patel, Prabhudas S

2009-01-01

Oral cancer is the leading malignancy in India. Nitric oxide and antioxidant enzymes play an important role in etiology of oral cancer. Therefore, the present study evaluates nitric oxide and antioxidant enzyme levels in healthy individual without tobacco habits (NHT, N=30) and healthy individuals with tobacco habits (WHT, n=90), patients with oral precancers (OPC, n=15) and oral cancer patients (n=126). Blood samples were collected from the subjects. NO2 + NO3 (nitrite+nitrate), superoxide dismutase (SOD) and catalase levels were estimated using highly specific spectrophotometeric methods. Statistical analysis was done by SPSS statistical software version 10. Mean plasma NO2 + NO3 levels were elevated in patients with OPC and oral cancer patients as compared to the controls. Mean activities of erythrocyte SOD and catalase were higher in WHT than NHT. Erythrocyte SOD and catalase levels were higher in WHT and patients with OPC as compared to NHT. The erythrocyte SOD and catalase activities were lower in oral cancer patients than patients with OPC. The erythrocyte SOD activity was higher in advanced oral cancer than the early disease. Erythrocyte catalase activity was lower in poorly differentiated tumors than well and moderately differentiated tumors. Pearson's correlation analysis revealed that alterations in plasma NO2 + NO3 levels were negatively associated with changes in erythrocyte SOD activities. The data revealed that the alterations in antioxidant activities were associated with production of nitric oxide in oral cancer, which may have significant role in oral carcinogenesis.

Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

NASA Astrophysics Data System (ADS)

Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil. In conclusion, earthworms contribute to the decomposition of carbohydrates through promoting enzyme activities involved in the C-cycle except for leucine aminopeptidase and cellobiohydrolase. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

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

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

PubMed Central

Shaltout, Hossam A.; Figueroa, Jorge P.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

2009-01-01

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension. PMID:19047579

Modification of deoiled cumin dietary fiber with laccase and cellulase under high hydrostatic pressure.

PubMed

Ma, Mengmei; Mu, Taihua

2016-01-20

In this study, we evaluated the effects of high hydrostatic pressure (HHP) and enzyme (laccase and cellulase) treatment on the structural, physicochemical, and functional properties and antioxidant activity of deoiled cumin dietary fiber (DF). HHP-enzyme treatment increased the contents of soluble dietary fiber (SDF) (30.37 g/100g), monosaccharides (except for glucose), uronic acids, and total polyphenol. HHP-enzyme treatment altered the honey-comb structure of DF and generated new polysaccharides. DF modified by HHP-enzyme treatment exhibited improved water retention capacity (10.02 g/g), water swelling capacity (11.19 mL/g), fat and glucose absorption capacities (10.44 g/g, 22.18-63.54 mmol/g), α-amylase activity inhibition ration (37.95%), and bile acid retardation index (48.85-52.58%). The antioxidant activity of DF was mainly correlated to total polyphenol content (R=0.8742). Therefore, DF modified by HHP-enzyme treatment from deoiled cumin could be used as a fiber-rich ingredient in functional foods. Copyright © 2015. Published by Elsevier Ltd.

REPRODUCTION AND AROMATASE ACTIVITY IN THE MARINE FISH CUNNER (TAUTOGOLABROUS ADSPERSUS) EXPOSED TO ATRAZINE AND OCTYLPHENOL IN THE LABORATORY

EPA Science Inventory

This study was conducted to test the hypothesis that reproduction in fish is altered by exposure to endocrine-disrupting chemicals (EDCs) that modify aromatase activity. Aromatase, a product of the CYP19 gene, is the enzyme that catalyzes the conversion of the androgens androst...

Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

PubMed

Khan, Sheeba; Priyamvada, Shubha; Khan, Sara A; Khan, Wasim; Farooq, Neelam; Khan, Farah; Yusufi, A N K

2009-07-01

Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

Soil microbial carbon utilization, enzyme activities and nutrient availability responses to Bidens pilosa and a non-invasive congener under different irradiances.

PubMed

Wei, Hui; Yan, Wenbin; Quan, Guoming; Zhang, Jiaen; Liang, Kaiming

2017-09-12

Two Bidens species (Bidens pilosa and B. bipinnata) that originate from America have been introduced widely in pan-tropics, with the former regarded as a noxious invasive weed whereas the latter naturalized as a plant resource. Whether the two species exhibit different effects on the belowground system remains rarely studied. This study was conducted to investigate soil microbial carbon (C) utilization, enzyme activities and available nitrogen, phosphorus and potassium contents under the two species in a subtropical garden soil of southern China under different levels of light intensity. Results showed that the microbial C utilization and enzyme activities were not significantly different under the two species, implying that the strong invasiveness of B. pilosa could not be due to the plant-soil microbe interactions, at least plant-induced alterations of microbial community function to utilize C substrates. Alternatively, available soil nitrogen and potassium contents were significantly higher under B. pilosa than under B. bipinnata in full sun, indicating that the strong invasiveness of B. pilosa could result from rapid nutrient mobilizations by B. pilosa. However, the differences turned non-significant as light intensity decreased, suggesting that light availability could substantially alter the plant effects on soil nutrient mobilizations.

Manganese inhibits mitochondrial aconitase: a mechanism of manganese neurotoxicity1

PubMed Central

Zheng, Wei; Ren, Sean; Graziano, Joseph H.

2014-01-01

The symptoms of Mn-induced neurotoxicity resemble those of Parkinson’s diseases. Since iron (Fe) appears to play a pivotal role in pathophysiology of Parkinson’s disease, we set out to test the hypothesis that alterations in Fe-requiring enzymes such as aconitase contribute to Mn-induced neurotoxicity. Mitochondrial fractions prepared from rat brain were preincubated with MnCl2 in vitro, followed by the enzyme assay. Mn treatment significantly inhibited mitochondrial aconitase activity (24% inhibition at 625 μM to 81% at 2.5 mM, p < 0.05). The inhibitory effect was reversible and Mn-concentration dependent, and was reversed by the addition of Fe (0.05–1 mM) to the reaction mixture. In an in vivo chronic Mn exposure model, rats received intraperitoneal injection of 6 mg/kg Mn as MnCl2 once daily for 30 consecutive days. Mn exposure led to a region-specific alteration in total aconitase (i.e., mitochondrial + cytoplasmic): 48.5% reduction of the enzyme activity in frontal cortex (p < 0.01), 33.7% in striatum (p < 0.0963), and 20.6% in substantia nigra (p < 0.139). Chronic Mn exposure increased Mn concentrations in serum, CSF, and brain tissues. The elevation of Mn in all selected brain regions (range between 3.1 and 3.9 fold) was similar in magnitude to that in CSF (3.1 fold) rather than serum (6.1 fold). The present results suggest that Mn alters brain aconitase activity, which may lead to the disruption of mitochondrial energy production and cellular Fe metabolism in the brain. PMID:9675333

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 fully assess our hypotheses.

Effect of water withdrawal on formation of free radical, proline accumulation and activities of antioxidant enzymes in ZAT12-transformed transgenic tomato plants.

PubMed

Chandra Rai, Avinash; Singh, Major; Shah, Kavita

2012-12-01

Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessive production alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C(2)H(2) type Zinc Finger Protein (ZFP) reported to control the expression of several stress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86 variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant and consistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increased proline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxide concentrations in the transformed tomato plants did not alter much, suggesting a well regulated formation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformed tomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation of osmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, the ZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regions affected with severe drought stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Sleep deprivation alters gene expression and antioxidant enzyme activity in mice splenocytes.

PubMed

Lungato, L; Marques, M S; Pereira, V G; Hix, S; Gazarini, M L; Tufik, S; D'Almeida, V

2013-03-01

Cellular defence against the formation of reactive oxygen species (ROS) involves a number of mechanisms in which antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) play an important role. The relation between sleep deprivation and oxidative stress has not yet been completely elucidated. Although some authors did not find evidence of this relationship, others found alterations in some oxidative stress markers in response to sleep deprivation. Thus, the objective of this study was to identify changes induced by sleep deprivation in the activity and gene expression of antioxidant enzymes in mice splenocytes, ideally corroborating a better understanding of the observed effects related to sleep deprivation, which could be triggered by oxidative imbalance. Splenocytes from mice sleep deprived for 72 h showed no significant difference in CAT and CuZnSOD gene expression compared with normal sleep mice. However, sleep-deprived mice did show higher MnSOD gene expression than the control group. Concerning enzymatic activity, CuZnSOD and MnSOD significantly increased after sleep deprivation, despite the expression in CuZnSOD remained unchanged. Moreover, CAT activity was significantly lower after sleep deprivation. The data suggest that the antioxidant system is triggered by sleep deprivation, which in turn could influence the splenocytes homoeostasis, thus interfering in physiological responses. © 2013 The Authors. Scandinavian Journal of Immunology © 2013 Blackwell Publishing Ltd.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

PubMed

Goswami, Moloy T; Chen, Guoan; Chakravarthi, Balabhadrapatruni V S K; Pathi, Satya S; Anand, Sharath K; Carskadon, Shannon L; Giordano, Thomas J; Chinnaiyan, Arul M; Thomas, Dafydd G; Palanisamy, Nallasivam; Beer, David G; Varambally, Sooryanarayana

2015-09-15

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Insights into intermolecular interactions, electrostatic properties and the stability of C646 in the binding pocket of p300 histone acetyltransferase enzyme: a combined molecular dynamics and charge density study.

PubMed

Sivanandam, Magudeeswaran; Saravanan, Kandasamy; Kumaradhas, Poomani

2017-10-30

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are enzymes that exhibit an important transcription activity. Dysfunction of these enzymes may lead to different diseases including cancer, cardiovascular, and other diseases. Therefore, these enzymes are the potential target for the generation of new therapeutics. C646 is a synthetic p300 HAT inhibitor; its structural and the electrostatic properties are the paradigm to understand its activity in the active site of p300 HAT enzyme. The docked C646 molecule in the active site forms expected key intermolecular interactions with the amino acid residues Trp1436, Tyr1467, and one water molecule (W1861); and these interactions are important for acetylation reaction. When compare the active site structure of C646 with the gas-phase structure, it is confirmed that the electron density distribution of polar bonds are highly altered, when the molecule present in the active site. In the gas-phase structure of C646, a large negative regions of electrostatic potential is found at the vicinity of O(4), O(5), and O(6) atoms; whereas, the negative region of these atoms are reduced in the active site. The molecular dynamics (MD) simulation also performed, it reveals the conformational stability and the intermolecular interactions of C646 molecule in the active site of p300.

Grape seed proanthocyanidins ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes: an in vivo study.

PubMed

Karthikeyan, K; Sarala Bai, B R; Niranjali Devaraj, S

2007-11-30

This study was designed to examine the effects of grape seed proanthocyanidins (GSP) against myocardial injury (MI) induced by isoproterenol (ISO), in a rat model. Induction of rats with ISO (85 mg/kg body weight, subcutaneously) for 2 days resulted in a significant decrease in the activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase). The activities of lysosomal enzymes (alpha-d-glucuronidase, alpha-d-N-acetylglucosaminidase, cathepsin-D, acid phosphatases and alpha-d-galactosidase) were increased significantly in the heart and serum of ISO-induced rats. The prior administration of GSP for 6 days a week for 5 weeks significantly increased the activities of mitochondrial and respiratory chain enzymes and significantly decreased the activities of lysosomal enzymes in the heart tissues of ISO-induced rats, which proves the stress stabilizing action of GSP. Oral administration of grape seed proanthocyanidins alone (50, 100 and 150 mg/kg) to normal rats did not show any significant effect in all the parameters studied. These biochemical functional alterations were supported by the macroscopic enzyme mapping assay of ischemic myocardium. Thus, this study shows that 100 and 150 mg/kg of GSP gives protection against ISO-induced MI and demonstrates that GSP has a significant effect in the protection of heart.

Modification of lignin content and composition in plants

DOEpatents

Ye, Zheng-Hua

2002-01-01

Plants and methods of preparing plants having reduced lignin content and/or altered lignin composition are provided. The activities of caffeoyl-CoA O-methyltransferase and/or caffeic acid O-methyltransferase enzymes in the modified plants are reduced.

Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes.

PubMed

Wang, Liya; Limongelli, Anna; Vila, Maya R; Carrara, Franco; Zeviani, Massimo; Eriksson, Staffan

2005-01-01

Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.

Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model

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

Dhawale, Vaibhav Shrirang; Amara, Venkateswara Rao

Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2 weeks. 48 h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocytemore » count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers. - Highlights: • Diminazene aceturate (DIZE), an ACE2 activator prevents ovalbumin-induced asthma. • DIZE acted by upregulating ACE2, downregulating ACE1, MAPKs, markers of inflammation, apoptosis. • DIZE reduced airway inflammation, fibrosis, right ventricular hypertrophy and restored airway responsiveness.« less

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.

Exposure of Mn and FeSODs, but not Cu/ZnSOD, to NO leads to nitrosonium and nitroxyl ions generation which cause enzyme modification and inactivation: an in vitro study.

PubMed

Niketíc, V; Stojanović, S; Nikolić, A; Spasić, M; Michelson, A M

1999-11-01

The effect of NO treatment in vitro on structural and functional alterations of Cu/Zn, Mn, and Fe type of SODs was studied. Significant difference in response to NO of Cu/ZnSOD compared to the Mn and Fe types was demonstrated. Cu/ZnSOD was shown to be stable with respect to NO: even on prolonged exposure, NO produced negligible effect on its structure and activity. In contrast, both Mn and Fe types were found to be NO-sensitive: exposure to NO led to their fast and extensive inactivation, which was accompanied by extensive structural alterations, including (in some of the samples tested) the cleavage of enzyme polypeptide chains, presumably at His residues of the enzyme metal binding sites. The generation of nitrosonium (NO+) and nitroxyl (NO-) ions in NO treated Mn and FeSODs, which produce enzyme modifications and inactivation, was demonstrated. The physiological and biomedical significance of described findings is briefly discussed.

Zinc Regulation of Aminopeptidase B Involved in Neuropeptide Production

PubMed Central

Hwang, Shin-Rong; Hook, Vivian

2009-01-01

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B. PMID:18571504

Dietary protein restriction causes modification in aluminum-induced alteration in glutamate and GABA system of rat brain

PubMed Central

Nayak, Prasunpriya; Chatterjee, Ajay K

2003-01-01

Background Alteration of glutamate and γ-aminobutyrate system have been reported to be associated with neurodegenerative disorders and have been postulated to be involved in aluminum-induced neurotoxicity as well. Aluminum, an well known and commonly exposed neurotoxin, was found to alter glutamate and γ-aminobutyrate levels as well as activities of associated enzymes with regional specificity. Protein malnutrition also reported to alter glutamate level and some of its metabolic enzymes. Thus the region-wise study of levels of brain glutamate and γ-aminobutyrate system in protein adequacy and inadequacy may be worthwhile to understand the mechanism of aluminum-induced neurotoxicity. Results Protein restriction does not have any significant impact on regional aluminum and γ-aminobutyrate contents of rat brain. Significant interaction of dietary protein restriction and aluminum intoxication to alter regional brain glutamate level was observed in the tested brain regions except cerebellum. Alteration in glutamate α-decarboxylase and γ-aminobutyrate transaminase activities were found to be significantly influenced by interaction of aluminum intoxication and dietary protein restriction in all the tested brain regions. In case of regional brain succinic semialdehyde content, this interaction was significant only in cerebrum and thalamic area. Conclusion The alterations of regional brain glutamate and γ-aminobutyrate levels by aluminum are region specific as well as dependent on dietary protein intake. The impact of aluminum exposure on the metabolism of these amino acid neurotransmitters are also influenced by dietary protein level. Thus, modification of dietary protein level or manipulation of the brain amino acid homeostasis by any other means may be an useful tool to find out a path to restrict amino acid neurotransmitter alterations in aluminum-associated neurodisorders. PMID:12657166

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune.

PubMed

Metreveli, Eka; Kachlishvili, Eva; Singer, Steven W; Elisashvili, Vladimir

2017-10-01

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis of pH-responsive β-CD-based star polymer and impact of its self-assembly behavior on pectinase activity.

PubMed

Hu, Dong; Yang, Hong; Liu, Jiangtao; Lei, Zhongli

2017-03-01

A novel type of pH-responsive star polymer based on β-cyclodextrin (β-CD) was synthesized and further covalently conjugated with enzyme. The impact of its self-assembly behavior on enzyme activity was investigated. In our design, azide containing the polymer (N 3 ) 7 -β-CD-(PtBA) 14 was synthesized via atom transfer radical polymerization of tert-butyl acrylate using (N 3 ) 7 -β-CD-(Br) 14 as the multifunctional initiator. The final product (N 3 ) 7 -β-CD-(PAA) 14 was obtained via hydrolysis and covalently conjugating pectinase onto pH-responsive polyacrylic acid (PAA) arms. PAA can change its conformation with the self-assembly by altered pH, leading its nanostructure into micellar nanoparticles in aqueous solution and further affecting the activity of immobilized pectinase. The results were proved by fluorescence spectroscopy and dynamic light scattering. This system proves that the activity of immobilized enzyme can be tailored predictably, and this pH-responsive polymer holds great potential for controllable delivery of enzymes. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Soil mineral alters the effect of Cd on the alkaline phosphatase activity.

PubMed

Tan, Xiangping; He, Yike; Wang, Ziquan; Li, Chenghui; Kong, Long; Tian, Haixia; Shen, Weijun; Megharaj, Mallavarapu; He, Wenxiang

2018-05-30

The toxicity of heavy metals (HMs) to soil enzymes is directly influenced by the status of the enzyme (free vs. immobilized on minerals) and the duration of exposure. However, little information is available on the interaction effect of HMs, mineral, and exposure time on soil enzyme activities. We investigated the interaction mechanism of alkaline phosphatase (ALP) with minerals (montmorillonite and goethite) and the response of free and immobilized ALP to cadmium (Cd) toxicity under different exposure times. The adsorption isotherms of ALP on both minerals were L-type. The maximum adsorption capacity of goethite for ALP was 3.96 times than montmorillonite, although both had similar adsorption constant (K). Goethite showed a greater inhibitory effect on ALP activity than montmorillonite. The toxicity of Cd to free- and goethite-ALP was enhanced with increasing exposure time, indicating a time-dependent inhibition. However, Cd toxicity to montmorillonite-ALP was not affected by the exposure time. The inhibition of Cd to soil enzyme activity is influenced by the properties of mineral complexes and the duration of exposure. A further understanding of the time pattern of HMs toxicity is helpful for accurately assessing the hazards of HMs to soil enzyme activity. Copyright © 2018 Elsevier Inc. All rights reserved.

A virus-based single-enzyme nanoreactor

NASA Astrophysics Data System (ADS)

Comellas-Aragonès, Marta; Engelkamp, Hans; Claessen, Victor I.; Sommerdijk, Nico A. J. M.; Rowan, Alan E.; Christianen, Peter C. M.; Maan, Jan C.; Verduin, Benedictus J. M.; Cornelissen, Jeroen J. L. M.; Nolte, Roeland J. M.

2007-10-01

Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or physically anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme experiments in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-molecule studies on their enzymatic behaviour. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution.

PubMed

Yonemoto, Isaac T; Matteri, Christopher W; Nguyen, Thao Amy; Smith, Hamilton O; Weyman, Philip D

2013-07-02

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide.

PubMed

Peters, J L; Castillo, F J; Heath, R L

1989-01-01

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.

Quercetin and rutin ameliorates sulphasalazine-induced spermiotoxicity, alterations in reproductive hormones and steroidogenic enzyme imbalance in rats.

PubMed

Osawe, S O; Farombi, E O

2018-06-01

Certain dietary flavonoids exhibit protective potentials against drug-induced male reproductive toxicities. We investigated the protective effects of quercetin and rutin on sulphasalazine-induced alterations in steroidogenic enzyme activity, hormone profile and spermiotoxicity in rats. Sulphasalazine (SASP, 600 mg/kg bw) was administered alone or in combination with quercetin (20 mg/kg bw) or rutin (10 mg/kg bw) for 14 days. SASP treatment significantly increased relative weights of the epididymis and seminal vesicles. Also, testicular and epididymal sperm numbers (TSN, ESN), motility, daily sperm production (DSP) and acrosome reaction (AR) significantly decreased. SASP altered plasma testosterone, luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels while testicular cholesterol levels, 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities were decreased. Elevated malondialdehyde levels and concomitant decrease in reduced glutathione, glutathione-S-transferase, peroxidase and superoxide dismutase activities were evident in testis and epididymis of SASP-treated rats. Quercetin or rutin co-treatment with SASP significantly reversed organ weights, preserved sperm integrity, restored plasma hormone levels and increased cholesterol levels, 3β-HSD and 17β-HSD activities in testis. Both flavonoids also prevented oxidative stress in testis and epididymis of SASP-treated rats. Quercetin and rutin protect against the negative effects of SASP treatment on reproductive capacity in male rats. © 2018 Blackwell Verlag GmbH.

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer’s mouse model

PubMed Central

Karuppagounder, Saravanan S.; Xu, Hui; Shi, Qingli; Chen, Lian H.; Pedrini, Steve; Pechman, David; Baker, Harriet; Beal, M. Flint; Gandy, Sam E.; Gibson, Gary E.

2009-01-01

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Aβ1–42 levels by about three-fold, β-CTF (C99) levels by 33% and β-secretase (BACE1) protein levels by 43%. TD induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Aβ and promotes accumulation of plaques independent of neuron loss or neuritic clusters. PMID:18406011

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.

Non-alcoholic fatty liver disease (NAFLD) - pathogenesis, classification, and effect on drug metabolizing enzymes and transporters.

PubMed

Cobbina, Enoch; Akhlaghi, Fatemeh

2017-05-01

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the presence of steatosis in more than 5% of hepatocytes with little or no alcohol consumption. Insulin resistance, the metabolic syndrome or type 2 diabetes and genetic variants of PNPLA3 or TM6SF2 seem to play a role in the pathogenesis of NAFLD. The pathological progression of NAFLD follows tentatively a "three-hit" process namely steatosis, lipotoxicity and inflammation. The presence of steatosis, oxidative stress and inflammatory mediators like TNF-α and IL-6 has been implicated in the alterations of nuclear factors such as CAR, PXR, PPAR-α in NAFLD. These factors may result in altered expression and activity of drug metabolizing enzymes (DMEs) or transporters. Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 is more consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas the activity of CYP2E1 and the efflux transporter MRP3 is up-regulated. However, it is not clear how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug variability in patients and could have serious implications for the safety and efficacy of xenobiotics. In this review, we summarize the effects of NAFLD on the regulation, expression and activity of major DMEs and transporters. We also discuss the potential mechanisms underlying these alterations.

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects.

PubMed

Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V

1997-07-20

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method.

PubMed

Martins, S; Karmali, A; Serralheiro, M L

2006-08-15

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

PubMed

Jámbrik, Katalin; Máthé, C; Vasas, G; Bácsi, I; Surányi, G; Gonda, S; Borbély, G; M-Hamvas, Márta

2010-01-01

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Cadmium neurotoxicity to a freshwater planarian.

PubMed

Wu, Jui-Pin; Lee, Hui-Ling; Li, Mei-Hui

2014-11-01

Although freshwater planarians are evolutionarily primitive, they are some of the simplest bilateral animals possessing integrated neural networks similar to those in vertebrates. We attempted to develop planarian Dugesia japonica as a model for investigating the neurotoxicity of environmental pollutants such as cadmium (Cd). This study was therefore designed to study the effects of Cd on the locomotor activity, neurobehavior, and neurological enzymes of D. japonica. After planarians were exposed to Cd at high concentrations, altered neurobehavior was observed that exhibited concentration-dependent patterns. Morphological alterations in Cd-treated planarians included irregular shape, body elongation, screw-like hyperkinesia, and bridge-like position. To study the direct effects of Cd on neurological enzymes, tissue homogenates of planarians were incubated in vitro with Cd before their activity was measured. Results showed that acetylcholinesterase (AChE), adenosine triphosphatase (ATPase), and monoamine oxidase A (MAO-A) activities were inhibited in a concentration-dependent manner. MAO-B activity was significantly induced by Cd at low concentrations and inhibited at high concentrations. Changes in the in vivo activity of AChE and ATPase were also found after planarians were treated with Cd at a sublethal concentration (5.56 μM). These observations indicate that neurotransmission systems in planarians are disturbed after Cd exposure.

Deletion of murine choline dehydrogenase results in diminished sperm motility.

PubMed

Johnson, Amy R; Craciunescu, Corneliu N; Guo, Zhong; Teng, Ya-Wen; Thresher, Randy J; Blusztajn, Jan K; Zeisel, Steven H

2010-08-01

Choline dehydrogenase (CHDH) catalyzes the conversion of choline to betaine, an important methyl donor and organic osmolyte. We have previously identified single nucleotide polymorphisms (SNPs) in the human CHDH gene that, when present, seem to alter the activity of the CHDH enzyme. These SNPs occur frequently in humans. We created a Chdh(-/-) mouse to determine the functional effects of mutations that result in decreased CHDH activity. Chdh deletion did not affect fetal viability or alter growth or survival of these mice. Only one of eleven Chdh(-/-) males was able to reproduce. Loss of CHDH activity resulted in decreased testicular betaine and increased choline and PCho concentrations. Chdh(+/+) and Chdh(-/-) mice produced comparable amounts of sperm; the impaired fertility was due to diminished sperm motility in the Chdh(-/-) males. Transmission electron microscopy revealed abnormal mitochondrial morphology in Chdh(-/-) sperm. ATP content, total mitochondrial dehydrogenase activity and inner mitochondrial membrane polarization were all significantly reduced in sperm from Chdh(-/-) animals. Mitochondrial changes were also detected in liver, kidney, heart, and testis tissues. We suggest that men who have SNPs in CHDH that decrease the activity of the CHDH enzyme could have decreased sperm motility and fertility.

Effect of aqueous extract of Tribulus terrestris on oxalate-induced oxidative stress in rats

PubMed Central

Kamboj, P.; Aggarwal, M.; Puri, S.; Singla, S. K.

2011-01-01

The present study was aimed at studying the effect of Tribulus terrestris on different parameters of oxidative stress and gene expression profiles of antioxidant enzymes in renal tissues of male wistar rats after induction of hyperoxaluria. The animals were divided into three groups. The animals in group I (control) were administered vehicle only. In group II, the animals were treated with ethylene glycol (hyperoxaluric agent) and those in group III were administered T. terrestris plant extract in addition to ethylene glycol. All treatments were continued for a period of seven weeks. Ethylene glycol feeding resulted in hyperoxaluria as well as increased excretion of calcium and phosphate. Serum creatinine, uric acid and blood urea nitrogen levels were also altered in hyperoxaluric animals. Various oxidative stress parameters viz. lipid peroxidation and activity of antioxidant enzymes were used to confirm the peroxidant state. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm whether steady-state transcription level of different antioxidant enzymes was altered. T. terrestris significantly reduced the excretion of oxalate, calcium, and phosphate along with decreased levels of blood urea nitrogen, uric acid and creatinine in serum. T. terrestris also reduced hyperoxaluria- caused oxidative stress, and restored antioxidant enzyme activity and their expression profile in kidney tissue. Histological analysis depicted that T. terrestris treatment decreased renal epithelial damage, inflammation, and restored normal glomerular morphology. PMID:21886973

Aromatase Activity in Sheepshead Minnow (Cyprinodon variegatus), Exposed to 17B-Trenbolone or 17B-estradiol in a Tier II Two-Generation Test

EPA Science Inventory

We tested the hypothesis that endocrine disrupting chemicals (EDCs) that alter fish reproduction will also modulate activity of the steroidogenic enzyme aromatase. There are two distinct isozymes of aromatase that have been characterized in fish, one predominating in brains and a...

Relevance of pharmacogenetic aspects of mercaptopurine metabolism in the treatment of interstitial lung disease.

PubMed

Bakker, Jaap A; Drent, Marjolein; Bierau, Jörgen

2007-09-01

Mercaptopurine therapy is increasingly important as immunosuppressive therapy in interstitial lung disease. We focus on human mercaptopurine metabolism and the defects in this metabolism causing adverse drug reactions. Defects in mercaptopurine metabolizing enzymes like thiopurine methyltransferase and inosine triphosphate pyrophosphohydrolase lead to severe adverse drug reactions, sometimes with fatal outcome. Other enzymes, still not thoroughly investigated, can give rise to toxic effects or decreased efficacy in mercaptopurine therapy when the activity of these enzymes is altered. Pharmacogenetic screening of potential patients for mercaptopurine therapy is important to avoid adverse drug reactions caused by inherited enzyme deficiencies in these metabolic pathways. Pretreatment screening for deficiencies of mercaptopurine metabolizing enzymes will significantly reduce the number of patients with an adverse drug reaction and concomitantly associated healthcare costs.

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

Krishnamurthy, Malathy; Hennelly, Scott Patrick; Dale, Taraka T.

The most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme catalyst. Until recently engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or by using one of several strategies to knock down or knock out a wasteful gene. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. We report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. Ourmore » design includes a cis-repressor at the 5’ end of the mRNA that forms a stem-loop helix occluding the ribosome binding site and blocking translation. An activating-RNA, expressed in trans, frees the RBS turning on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability followed by directed evolution on a portion of each trans-activator to fine tune translation. We report a cis-repressor that can completely shut off translation of antibiotic resistance reporters and a trans-activator that restores translation. We have shown it is possible to use riboregulators to achieve translational control of gene expression over a wide dynamic range. Using a bioluminescent reporter system, we demonstrated an ON/OFF ratio >300. We have demonstrated that a targeting sequence can be changed to develop riboregulators that can independently regulate translation of many genes with minimal cross-talk. In a SELEX experiment, we demonstrated that by subtly altering the sequence of the trans-activator, it is possible to alter the equilibrium between repressed and activated states and achieve intermediate translational control.« less

Tunable riboregulator switches for post-transcriptional control of gene expression

DOE PAGES

Krishnamurthy, Malathy; Hennelly, Scott Patrick; Dale, Taraka T.; ...

2015-07-13

The most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme catalyst. Until recently engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or by using one of several strategies to knock down or knock out a wasteful gene. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. We report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. Ourmore » design includes a cis-repressor at the 5’ end of the mRNA that forms a stem-loop helix occluding the ribosome binding site and blocking translation. An activating-RNA, expressed in trans, frees the RBS turning on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability followed by directed evolution on a portion of each trans-activator to fine tune translation. We report a cis-repressor that can completely shut off translation of antibiotic resistance reporters and a trans-activator that restores translation. We have shown it is possible to use riboregulators to achieve translational control of gene expression over a wide dynamic range. Using a bioluminescent reporter system, we demonstrated an ON/OFF ratio >300. We have demonstrated that a targeting sequence can be changed to develop riboregulators that can independently regulate translation of many genes with minimal cross-talk. In a SELEX experiment, we demonstrated that by subtly altering the sequence of the trans-activator, it is possible to alter the equilibrium between repressed and activated states and achieve intermediate translational control.« less

Subacute exposure to N-ethyl perfluorooctanesulfonamidoethanol results in the formation of perfluorooctanesulfonate and alters superoxide dismutase activity in female rats.

PubMed

Xie, Wei; Wu, Qian; Kania-Korwel, Izabela; Tharappel, Job C; Telu, Sanjay; Coleman, Mitchell C; Glauert, Howard P; Kannan, Kurunthachalam; Mariappan, S V S; Spitz, Douglas R; Weydert, Jamie; Lehmler, Hans-Joachim

2009-10-01

Perfluorooctanesulfonamides, such as N-ethyl perfluorooctanesulfonamidoethanol (N-EtFOSE), are large scale industrial chemicals but their disposition and toxicity are poorly understood despite significant human exposure. The hypothesis that subacute exposure to N-EtFOSE, a weak peroxisome proliferator, causes a redox imbalance in vivo was tested using the known peroxisome proliferator, ciprofibrate, as a positive control. Female Sprague-Dawley rats were treated orally with N-EtFOSE, ciprofibrate or corn oil (vehicle) for 21 days, and levels of N-EtFOSE and its metabolites as well as markers of peroxisome proliferation and oxidative stress were assessed in serum, liver and/or uterus. The N-EtFOSE metabolite profile in liver and serum was in good agreement with reported in vitro biotransformation pathways in rats and the metabolite levels decreasing in the order perfluorooctanesulfonate > perfluorooctanesulfonamide ~ N-ethyl perfluorooctanesulfonamidoacetate > perfluorooctanesulfonamidoethanol approximately N-EtFOSE. Although N-EtFOSE treatment significantly decreased the growth rate, increased relative liver weight and activity of superoxide dismutases (SOD) in liver and uterus (total SOD, CuZnSOD and MnSOD), a metabolic study revealed no differences in the metabolome in serum from N-EtFOSE-treated and control animals. Ciprofibrate treatment increased liver weight and peroxisomal acyl Co-A oxidase activity in the liver and altered antioxidant enzyme activities in the uterus and liver. According to NMR metabolomic studies, ciprofibrate treated animals had altered serum lipid profiles compared to N-EtFOSE-treated and control animals, whereas putative markers of peroxisome proliferation in serum were not affected. Overall, this study demonstrates the biotransformation of N-EtFOSE to PFOS in rats that is accompanied by N-EtFOSE-induced alterations in antioxidant enzyme activity.

Subacute Exposure to N-Ethyl Perfluorooctanesulfonamidoethanol Results in the Formation of Perfluorooctanesulfonate and Alters Superoxide Dismutase Activity in Female Rats

PubMed Central

Xie, Wei; Wu, Qian; Kania-Korwel, Izabela; Tharappel, Job C.; Telu, Sanjay; Coleman, Mitchell C.; Glauert, Howard P.; Kannan, Kurunthachalam; Santhana Mariappan, S. V.; Spitz, Douglas R.; Weydert, Jamie; Lehmler, Hans-Joachim

2009-01-01

Perfluorooctanesulfonamides, such as N-ethyl perfluorooctanesulfonamidoethanol (N-EtFOSE), are large scale industrial chemicals but their disposition and toxicity are poorly understood despite significant human exposure. The hypothesis that subacute exposure to N-EtFOSE, a weak peroxisome proliferator, causes a redox imbalance in vivo was tested using the known peroxisome proliferator, ciprofibrate, as a positive control. Female Sprague-Dawley rats were treated orally with N-EtFOSE, ciprofibrate or corn oil (vehicle) for 21 days, and levels of N-EtFOSE and its metabolites as well as markers of peroxisome proliferation and oxidative stress were assessed in serum, liver and/or uterus. The N-EtFOSE metabolite profile in liver and serum was in good agreement with reported in vitro biotransformation pathways in rats and the metabolite levels decreasing in the order perfluorooctanesulfonate ≫ perfluorooctanesulfonamide ∼ N-ethyl perfluorooctanesulfonamidoacetate ≫ perfluorooctanesulfonamidoethanol ∼ N-EtFOSE. Although N-EtFOSE treatment significantly decreased the growth rate, increased relative liver weight and activity of superoxide dismutases (SOD) in liver and uterus (total SOD, CuZnSOD and MnSOD), a metabolic study revealed no differences in the metabolome in serum from N-EtFOSE-treated and control animals. Ciprofibrate treatment increased liver weight and peroxisomal acyl Co-A oxidase activity in the liver and altered antioxidant enzyme activities in the uterus and liver. According to NMR metabolomic studies, ciprofibrate treated animals had altered serum lipid profiles compared to N-EtFOSE-treated and control animals, whereas putative markers of peroxisome proliferation in serum were not affected. Overall, this study demonstrates the biotransformation of N-EtFOSE to PFOS in rats that is accompanied by N-EtFOSE-induced alterations in antioxidant enzyme activity. PMID:19544052

Network of proteins, enzymes and genes linked to biomass degradation shared by Trichoderma species.

PubMed

Horta, Maria Augusta Crivelente; Filho, Jaire Alves Ferreira; Murad, Natália Faraj; de Oliveira Santos, Eidy; Dos Santos, Clelton Aparecido; Mendes, Juliano Sales; Brandão, Marcelo Mendes; Azzoni, Sindelia Freitas; de Souza, Anete Pereira

2018-01-22

Understanding relationships between genes responsible for enzymatic hydrolysis of cellulose and synergistic reactions is fundamental for improving biomass biodegradation technologies. To reveal synergistic reactions, the transcriptome, exoproteome, and enzymatic activities of extracts from Trichoderma harzianum, Trichoderma reesei and Trichoderma atroviride under biodegradation conditions were examined. This work revealed co-regulatory networks across carbohydrate-active enzyme (CAZy) genes and secreted proteins in extracts. A set of 80 proteins and respective genes that might correspond to a common system for biodegradation from the studied species were evaluated to elucidate new co-regulated genes. Differences such as one unique base pair between fungal genomes might influence enzyme-substrate binding sites and alter fungal gene expression responses, explaining the enzymatic activities specific to each species observed in the corresponding extracts. These differences are also responsible for the different architectures observed in the co-expression networks.

Measurement of Enzyme Isotope Effects.

PubMed

Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

2017-01-01

Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

PubMed Central

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

2014-01-01

ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses. PMID:24600002

The crystal structure of a cardiovirus RNA-dependent RNA polymerase reveals an unusual conformation of the polymerase active site.

PubMed

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J M; Ferrer-Orta, Cristina; Verdaguer, Núria

2014-05-01

Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Effect of elevated CO2 on the interaction between invasive thrips, Frankliniella occidentalis, and its host kidney bean, Phaseolus vulgaris.

PubMed

Qian, Lei; He, Shuqi; Liu, Xiaowei; Huang, Zujin; Chen, Fajun; Gui, Furong

2018-05-08

Elevated CO 2 can alter the leaf damage caused by insect herbivores. Frankliniella occidentalis (Pergande) is highly destructive invasive pest in crop production worldwide. To investigate how elevated CO 2 affects F. occidentalis fed with Phaseolus vulgaris and in particularly, the interaction between plant defense and thrips anti-defense, nutrients content and antioxidant enzymes activity of P. vulgaris have been measured, as well as the detoxifying enzymes activity of adult thrips. Elevated CO 2 increased soluble sugar, soluble protein, and free amino acids content in non-thrips plants, and decreased SOD and POD activity in these plants. Thrips feeding reduced the nutrients content in plants, and increased their SOD, CAT and POD activity. Variation of nutrients content and antioxidant enzymes activity in plants showed an opposite tendency over thrips feeding time. After feeding, AchE, CarE, and MFO activity in thrips increased to against plant defense. More thrips densities induced stronger plant defense, in return, detoxifying enzymes in thrips increased over thrips number. Our study revealed that F. occidentalis can induce not only antioxidant-associated plant defense, but also the thrips detoxifying enzymes. Elevated CO 2 might not only enhance plant defense to thrips attack, but also increase thrips anti-defense against plant defense. This article is protected by copyright. All rights reserved.

Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L

PubMed Central

2010-01-01

Background Increased Al concentration causes reduction of mitotic activity, induction of nucleolar alteration, increase of the production of ROS and alteration of several antioxidant enzyme activities in plant cells. Allium cepa is an excellent plant and a useful biomarker for environmental monitoring. Limited information is available about the effects of Al on nucleoli, antioxidant enzyme system, contents of MDA and soluble protein in A. cepa. Therefore, we carried out the investigation in order to better understand the effects of Al on the growth, nucleoli in root tip cells and selected physiological and biochemical characters. Results The results showed that the root growth exposed to 50 μM Al was inhibited significantly. 50 μM Al could induce some particles of argyrophilic proteins scattered in the nuclei and extruded from the nucleoli into the cytoplasm. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar reconstruction was inhibited. 50 μM Al induced high activities of SOD and POD in leaves and roots significantly (P < 0.05) when compared with control, whereas the level of CAT was low significantly (P < 0.05). At 50 μM Al the content of MDA in leaves was high significantly (P < 0.05) at 9th day and in roots increased (P < 0.05) with prolonging the treatment time during 6-12 days. The soluble protein content in leaves treated with 50 μM Al was high significantly (P < 0.05) at 6th day and increased with prolonging the treatment time. Conclusions We suggest that variations in nucleoli and the alterations of antioxidant enzyme activities, MDA and soluble protein contents in Allium cepa can serve as useful biomarkers, which can provide valuable information for monitoring and forecasting effects of exposure to Al in real scenarios conditions. Among the antioxidant enzymes SOD and POD appear to play a key role in the antioxidant defense mechanism under Al toxicity condition. Data from MDA concentration show that Al indirectly produces superoxide radicals, resulting in increased lipid peroxidative products and oxidative stress. PMID:20964828

Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L.

PubMed

Qin, Rong; Jiao, Yunqiu; Zhang, Shanshan; Jiang, Wusheng; Liu, Donghua

2010-10-21

Increased Al concentration causes reduction of mitotic activity, induction of nucleolar alteration, increase of the production of ROS and alteration of several antioxidant enzyme activities in plant cells. Allium cepa is an excellent plant and a useful biomarker for environmental monitoring. Limited information is available about the effects of Al on nucleoli, antioxidant enzyme system, contents of MDA and soluble protein in A. cepa. Therefore, we carried out the investigation in order to better understand the effects of Al on the growth, nucleoli in root tip cells and selected physiological and biochemical characters. The results showed that the root growth exposed to 50 μM Al was inhibited significantly. 50 μM Al could induce some particles of argyrophilic proteins scattered in the nuclei and extruded from the nucleoli into the cytoplasm. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar reconstruction was inhibited. 50 μM Al induced high activities of SOD and POD in leaves and roots significantly (P < 0.05) when compared with control, whereas the level of CAT was low significantly (P < 0.05). At 50 μM Al the content of MDA in leaves was high significantly (P < 0.05) at 9(th) day and in roots increased (P < 0.05) with prolonging the treatment time during 6-12 days. The soluble protein content in leaves treated with 50 μM Al was high significantly (P < 0.05) at 6(th) day and increased with prolonging the treatment time. We suggest that variations in nucleoli and the alterations of antioxidant enzyme activities, MDA and soluble protein contents in Allium cepa can serve as useful biomarkers, which can provide valuable information for monitoring and forecasting effects of exposure to Al in real scenarios conditions. Among the antioxidant enzymes SOD and POD appear to play a key role in the antioxidant defense mechanism under Al toxicity condition. Data from MDA concentration show that Al indirectly produces superoxide radicals, resulting in increased lipid peroxidative products and oxidative stress.

Epigenetic alterations are associated with monocyte immune dysfunctions in HIV-1 infection.

PubMed

Espíndola, Milena S; Soares, Luana S; Galvão-Lima, Leonardo J; Zambuzi, Fabiana A; Cacemiro, Maira C; Brauer, Verônica S; Marzocchi-Machado, Cleni M; de Souza Gomes, Matheus; Amaral, Laurence R; Martins-Filho, Olindo A; Bollela, Valdes R; Frantz, Fabiani G

2018-04-03

Monocytes are key cells in the immune dysregulation observed during human immunodeficiency virus (HIV) infection. The events that take place specifically in monocytes may contribute to the systemic immune dysfunction characterized by excessive immune activation in infected individuals, which directly correlates with pathogenesis and progression of the disease. Here, we investigated the immune dysfunction in monocytes from untreated and treated HIV + patients and associated these findings with epigenetic changes. Monocytes from HIV patients showed dysfunctional ability of phagocytosis and killing, and exhibited dysregulated cytokines and reactive oxygen species production after M. tuberculosis challenge in vitro. In addition, we showed that the expression of enzymes responsible for epigenetic changes was altered during HIV infection and was more prominent in patients that had high levels of soluble CD163 (sCD163), a newly identified plasmatic HIV progression biomarker. Among the enzymes, histone acetyltransferase 1 (HAT1) was the best epigenetic biomarker correlated with HIV - sCD163 high patients. In conclusion, we confirmed that HIV impairs effector functions of monocytes and these alterations are associated with epigenetic changes that once identified could be used as targets in therapies aiming the reduction of the systemic activation state found in HIV patients.

Alteration of extracellular enzyme activity and microbial abundance by biochar addition: Implication for carbon sequestration in subtropical mangrove sediment.

PubMed

Luo, Ling; Gu, Ji-Dong

2016-11-01

Biochar has attracted more and more attention due to its essential role in adsorbing pollutants, improving soil fertility, and modifying greenhouse gas emission. However, the influences of biochar on extracellular enzyme activity and microbial abundance are still lack and debatable. Currently, there is no information about the impact of biochar on the function of mangrove ecosystems. Therefore, we explored the effects of biochar on extracellular enzyme activity and microbial abundance in subtropical mangrove sediment, and further estimated the contribution of biochar to C sequestration. In this study, sediments were amended with 0 (control), 0.5, 1.0 and 2.0% of biochar and incubated at 25 °C for 90 days. After incubation, enzyme activities, microbial abundance and the increased percentage of sediment organic C content were determined. Both increase (phenol oxidase and β-glucosidase) and decrease (peroxidase, N-acetyl-glucosaminidase and acid phosphatase) of enzyme activities were observed in biochar treatments, but only peroxidase activity showed statistical significance (at least p < 0.01) compared to the control. Moreover, the activities of all enzymes tested were significantly related to the content of biochar addition (at least p < 0.05). On the other hand, bacterial and fungal abundance in biochar treatments were remarkably lower than control (p < 0.001), and the significantly negative relationship (p < 0.05) between bacterial abundance and the content of biochar was found. Additionally, the increased percentage of organic C gradually increased with biochar addition rate, which provided evidence for applying biochar to mitigate climate change. Given the importance of microorganisms and enzyme activities in sediment organic matter decomposition, the increased C sequestration might be explained by the large decrease of microbial abundance and enzyme activity after biochar intervention. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nanoparticle bioconjugates as "bottom-up" assemblies of artifical multienzyme complexes

NASA Astrophysics Data System (ADS)

Keighron, Jacqueline D.

2010-11-01

The sequential enzymes of several metabolic pathways have been shown to exist in close proximity with each other in the living cell. Although not proven in all cases, colocalization may have several implications for the rate of metabolite formation. Proximity between the sequential enzymes of a metabolic pathway has been proposed to have several benefits for the overall rate of metabolite formation. These include reduced diffusion distance for intermediates, sequestering of intermediates from competing pathways and the cytoplasm. Restricted diffusion in the vicinity of an enzyme can also cause the pooling of metabolites, which can alter reaction equilibria to control the rate of reaction through inhibition. Associations of metabolic enzymes are difficult to isolate ex vivo due to the weak interactions believed to colocalize sequential enzymes within the cell. Therefore model systems in which the proximity and diffusion of intermediates within the experiment system are controlled are attractive alternatives to explore the effects of colocalization of sequential enzymes. To this end three model systems for multienzyme complexes have been constructed. Direct adsorption enzyme:gold nanoparticle bioconjugates functionalized with malate dehydrogenase (MDH) and citrate synthase (CS) allow for proximity between to the enzymes to be controlled from the nanometer to micron range. Results show that while the enzymes present in the colocalized and non-colocalized systems compared here behaved differently overall the sequential activity of the pathway was improved by (1) decreasing the diffusion distance between active sites, (2) decreasing the diffusion coefficient of the reaction intermediate to prevent escape into the bulk solution, and (3) decreasing the overall amount of bioconjugate in the solution to prevent the pathway from being inhibited by the buildup of metabolite over time. Layer-by-layer (LBL) assemblies of MDH and CS were used to examine the layering effect of sequential enzymes found in multienzyme complexes such as the pyruvate dehydrogenase complex (PDC). By controlling the orientation of enzymes in the complex (i.e. how deeply embedded each enzyme is) it was hypothesized that differences in sequential activity would determine an optimal orientation for a multienzyme complex. It was determined during the course of these experiments that the polyelectrolyte (PE) assembly itself served to slow diffusion of intermediates, leading to a buildup of oxaloacetate within the PE layers to form a pool of metabolite that equalized the rate of sequential reaction between the different orientations tested. Hexahistidine tag -- Ni(II) nitriliotriacetic acid (NTA) chemistry is an attractive method to control the proximity between sequential enzymes because each enzyme can be bound in a specific orientation, with minimal loss of activity, and the interaction is reversible. Modifying gold nanoparticles or large unilamellar vesicles with this functionality allows for another class of model to be constructed in which proximity between enzymes is dynamic. Some metabolic pathways (such as the de novo purine biosynthetic pathway), have demonstrated dynamic proximity of sequential enzymes in response to specific cellular stimuli. Results indicate that Ni(II)NTA scaffolds immobilize histidine-tagged enzymes non-destructively, with a near 100% reversibility. This model can be used to demonstrate the possible implications of dynamic proximity such as pathway regulation. Insight into the benefits and mechanisms of sequential enzyme colocalization can enhance the general understanding of cellular processes, as well as allow for the development of new and innovative ways to modulate pathway activity. This may provide new designs for treatments of metabolic diseases and cancer, where metabolic pathways are altered.

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions.

PubMed

Hsiao, Ju-Chun; McGrath, Aaron P; Kielmann, Linda; Kalimuthu, Palraj; Darain, Farzana; Bernhardt, Paul V; Harmer, Jeffrey; Lee, Mihwa; Meyers, Kimberley; Maher, Megan J; Kappler, Ulrike

2018-01-01

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo VI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT WT , precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of polystyrene microbeads in marine planktonic crustaceans.

PubMed

Gambardella, Chiara; Morgana, Silvia; Ferrando, Sara; Bramini, Mattia; Piazza, Veronica; Costa, Elisa; Garaventa, Francesca; Faimali, Marco

2017-11-01

Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10mgL -1 ) for 24 and 48h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1mgL -1 ) after 48h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative Enzymatic and Immunologic Histophotometry of Diseased Human Kid-Ney Tissues Using Tv-Camera and Computer Assisted Image Processing Systems.

NASA Astrophysics Data System (ADS)

Heinert, G.; Mondorf, W.

1982-11-01

High speed image processing was used to analyse morphologic and metabolic characteristics of clinically relevant kidney tissue alterations.Qualitative computer-assisted histophotometry was performed to measure alterations in levels of the enzymes alkaline phosphatase (Ap),alanine aminopeptidase (AAP),g-glutamyltranspepti-dase (GGTP) and A-glucuronidase (B-G1) and AAP and GGTP immunologically determined in prepared renal and cancer tissue sections. A "Mioro-Videomat 2" image analysis system with a "Tessovar" macroscope,a computer-assisted "Axiomat" photomicroscope and an "Interactive Image Analysis System (IBAS)" were employed for analysing changes in enzyme activities determined by changes in absorbance or transmission.Diseased kidney as well as renal neoplastic tissues could be distinguished by significantly (wilcoxon test,p<0,05) decreased enzyme concentrations as compared to those found in normal human kidney tissues.This image analysis techniques might be of potential use in diagnostic and prognostic evaluation of renal cancer and diseased kidney tissues.

S-allylcysteine ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes.

PubMed

Padmanabhan, M; Mainzen Prince, P Stanely

2007-02-13

This study was aimed to evaluate the preventive role of S-allylcysteine (SAC) on mitochondrial and lysosomal enzymes in isoproterenol (ISO)-induced rats. Male albino Wistar rats were pretreated with SAC (50, 100 and 150 mg/kg) daily for a period of 45 days. After the treatment period, ISO (150 mg/kg) was subcutaneously injected to rats at an interval of 24 h for two days. The activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome C oxidase) were decreased significantly (p<0.05) in ISO-induced rats. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-D and acid phosphatase) were increased significantly (p<0.05) in serum and heart of ISO-induced rats. Pretreatment with SAC (100 mg/kg and 150 mg/kg) for a period of 45 days increased significantly (p<0.05) the activities of mitochondrial and respiratory chain enzymes and decreased the activities of lysosomal enzymes significantly (p<0.05) in ISO-induced rats. Oral administration of SAC (50, 100 and 150 mg/kg) for a period of 45 days to normal rats did not show any significant (p<0.05) effect in all the parameters studied. The altered electrocardiogram (ECG) of ISO-treated rats was also restored to near normal by treatment with SAC (100 and 150 mg/kg). These results confirm the efficacy of SAC in alleviating ISO-induced cardiac damage.

E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

PubMed

Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

2015-11-01

In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

Effect of acute hypoxic shock on the rat brain morphology and tripeptidyl peptidase I activity.

PubMed

Petrova, Emilia B; Dimitrova, Mashenka B; Ivanov, Ivaylo P; Pavlova, Velichka G; Dimitrova, Stella G; Kadiysky, Dimitar S

2016-06-01

Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress. Copyright © 2016 Elsevier GmbH. All rights reserved.

The role of strong electrostatic interactions at the dimer interface of human glutathione synthetase.

PubMed

De Jesus, Margarita C; Ingle, Brandall L; Barakat, Khaldoon A; Shrestha, Bisesh; Slavens, Kerri D; Cundari, Thomas R; Anderson, Mary E

2014-10-01

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein-protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.

Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

PubMed Central

Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

2015-01-01

Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development

PubMed Central

Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

2015-01-01

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823

Role of the adverse outcome pathway framework in the validation of predictive biomarkers

EPA Science Inventory

Gene expression, enzyme activities, changes in endogenous metabolite or hormone titers, altered histology, etc. are widely used as biomarkers, but rarely, if ever, used for regulatory decision-making or to define management objectives. The disconnect between the measurements comm...

Cardioprotective activity of flax lignan concentrate extracted from seeds of Linum usitatissimum in isoprenalin induced myocardial necrosis in rats

PubMed Central

Zanwar, Anand A.; Hegde, Mahabaleshwar V.; Bodhankar, Subhash L.

2011-01-01

The objective of the study was to evaluate the cardioprotective activity of flax lignan concentrate (FLC) in isoprenalin (ISO) induced cardiotoxicity in rats. Male Wistar rats (200–230 g) were divided into three groups. Group I: control, Group II: isoprenalin, Group III: FLC (500 mg/kg, p.o.) orally for 8 days and in group II and III isoprenalin 5.25 mg/kg, s.c. on day 9 and 8.5 mg/kg on day 10. On day 10 estimation of marker enzymes in serum and haemodynamic parameters were recorded. Animals were sacrificed, histology of heart was performed. Isoprenalin showed cardiotoxicity, manifested by increased levels of marker enzymes and increased heart rate. FLC treatment reversed these biochemical changes significantly compared with ISO group. The cardiotoxic effect of isoprenalin was less in FLC pretreated animals, which was confirmed in histopathological alterations. Haemodynamic, biochemical alteration and histopathological results suggest a cardioprotective protective effect of FLC in isoprenalin induced cardiotoxicity. PMID:21753905

Serum concentrations of trace elements and their relationships with paraoxonase-1 in morbidly obese women.

PubMed

Luciano-Mateo, Fedra; Cabré, Noemí; Nadal, Martí; García-Heredia, Anabel; Baiges-Gaya, Gerard; Hernández-Aguilera, Anna; Camps, Jordi; Joven, Jorge; Domingo, José Luis

2018-07-01

The metabolic alterations associated with obesity include mineral dysregulation. Essential trace elements are nutrients with a relevant function in a large number of cellular processes and multiple roles in the correct functioning of metabolic enzymes. Paraoxonase-1 (PON1) is an antioxidant and anti-inflammatory enzyme that is compromised in obesity. In the present study, the potential alterations in trace elements in morbidly obese women were assessed in relation to serum PON1 activity and concentration, as well as to other obesity-related comorbidities such as diabetes mellitus and fatty liver. We recruited 41 morbidly obese women and 51 control individuals. The serum concentrations of 30 elements, PON1 paraoxonase and lactonase activities, and PON1 concentration were measured. We observed significant alterations in the levels of As, Ba, Cu, Ca, Fe, Mg, Na, Se, Sr, and Zn in obese women; some of them (As, Ca, Cr, Cu, Mg, and Se) being significantly correlated with serum PON1 values. The most relevant changes were observed in the concentrations of As, Sr and Mg, the last of which was also significantly associated with diabetes mellitus. The current results raise the possibility that increased ingestion and/or storage of a number of trace elements may be factors predisposing to obesity-related comorbidities and metabolic alterations. Copyright © 2018 Elsevier GmbH. All rights reserved.

Hematoporphyrin derivative induced photodamage to brain tumor cells: Alterations in subcellular membranes

NASA Astrophysics Data System (ADS)

Sreenivasan, Rajesh; Joshi, Preeti G.; Joshi, Nanda B.

1997-01-01

Photoinduced structural and functional changes were studied in the subcellular membranes isolated from HpD treated cells. Changes in the limiting anisotropy of lipid specific probes 1,6,Diphenyl-1,3,5,hexatriene (DPH) and 1-(4-Trimethyl ammonium 1,6 diphenyl)-1,3,5,hexatriene toulene sulphonate (TMA-DPH) incorporated into the membrane were used to assess the structural alterations while changes in the activity of the marker enzymes were used to assess the functional alterations. Our results suggest that damage to the endoplasmic reticulum may play an important role in the photosensitization of brain tumor cells.

Calcium signaling in plant cells in altered gravity

NASA Astrophysics Data System (ADS)

Kordyum, E. L.

2003-10-01

Changes in the intracellular Ca 2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca 2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80 th, a review highlighting the performed research and the possible significance of such Ca 2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension → alterations in the physicochemical properties of the membrane → changes in membrane permeability, ion transport, membrane-bound enzyme activity, etc. → metabolism rearrangements → physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca 2+ messenger system. Changes in Ca 2+ influx/efflux and possible pathways of Ca 2+ signaling in plant cell biochemical regulation in altered gravity are discussed.

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

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

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE PAGES

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; ...

2017-06-14

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

PubMed

Stock, Roberto P; Brewer, Jonathan; Wagner, Kerstin; Ramos-Cerrillo, Blanca; Duelund, Lars; Jernshøj, Kit Drescher; Olsen, Lars Folke; Bagatolli, Luis A

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily*

PubMed Central

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C.; Yew, Wen Shan

2010-01-01

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of kcat/Km of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis. PMID:20980257

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

PubMed

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C; Yew, Wen Shan

2010-12-24

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

Stachybotrys chartarum alters surfactant-related phospholipid synthesis and CTP:cholinephosphate cytidylyltransferase activity in isolated fetal rat type II cells.

PubMed

Hastings, C; Rand, T; Bergen, H T; Thliveris, J A; Shaw, A R; Lombaert, G A; Mantsch, H H; Giles, B L; Dakshinamurti, S; Scott, J E

2005-03-01

Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.

Binding of Cimetidine to Balb/C Mouse Liver Catalase; Kinetics and Conformational Studies.

PubMed

Jahangirvand, Mahboubeh; Minai-Tehrani, Dariush; Yazdi, Fatemeh; Minai-Tehrani, Arash; Razmi, Nematollah

2016-01-01

Catalase is responsible for converting hydrogen peroxide (H2O2) into water and oxygen in cells. This enzyme has high affinity for hydrogen peroxide and can protect the cells from oxidative stress damage. Catalase is a tetramer protein and each monomer contains a heme group. Cimetidine is a histamine H2 receptor blocker which inhibits acid release from stomach and is used for gasterointestinal diseases. In this research, effect of cimetidine on the activity of liver catalase was studied and the kinetic parameters of this enzyme and its conformational changes were investigated. Cell free extract of mouse liver was used for the catalase assay. The activity of the catalase was detected in the absence and presence of cimetidine by monitoring hydrogen peroxide reduction absorbance at 240 nm. The purified enzyme was used for conformational studies by Fluorescence spectrophotometry. The data showed that cimetidine could inhibit the enzyme in a non-competitive manner. Ki and IC50 values of the drug were determined to be about 0.75 and 0.85 uM, respectively. The Arrhenius plot showed that activation energy was 6.68 and 4.77 kJ/mol in the presence and absence of the drug, respectively. Fluorescence spectrophotometry revealed that the binding of cimetidine to the purified enzyme induced hyperchromicity and red shift which determined the conformational change on the enzyme. Cimetidine could non-competitively inhibit the liver catalase with high affinity. Binding of cimetidine to the enzyme induced conformational alteration in the enzyme.

A six-year longitudinal study of phosphorus enrichment on soil enzymes in acidic forest soils.

NASA Astrophysics Data System (ADS)

Deforest, J. L.; Freedman, Z.

2017-12-01

Acidic nitrogen (N) deposition may be shifting the nutrient economies of forest soils from one dominated by N more towards phosphorus (P) limitation. While the short-term responses of nutrient enrichment experiments are reported, there is a lack of information on the longer-term response mediating ecosystem nutrient dynamics, especially for P. We hypothesized that long-term soil P amendments should result in the persistent suppression of P-acquiring extracellular enzymes when compared with ambient soils. Alternatively, vegetation and/or the microbial community may have acclimated to require more P (i.e., communities more suitable to the altered nutrient economy) resulting in an increase in the activity of P-acquiring enzymes relative to carbon (C) and N-acquiring enzyme activity. To test the hypothesis, P availability was indirectly and/or directly increased by raising soil pH and/or the addition of phosphate fertilizer and maintained for six years. Study sites were in two North American eastern deciduous forest regions on glaciated soils with modest P availability and unglaciated with low P availability. For the glaciated sites, C:N acquiring enzyme activity remained stable and was insensitive to 6 years of elevated pH and/or P in the, but there was modest increases in the unglaciated site. Phosphorus-acquiring enzyme activity was insensitive to the treatments in the glaciated sites. For unglaciated sites, P-acquiring enzyme activity was suppressed under P addition in year one, rebounded in the second year, and was suppressed in the subsequent years. These results suggest that the basal nutrient resources of an ecosystem will have a very strong influence on its response to nutrient enrichment. Likewise, the second-year recovery of P-acquiring enzyme activity might be evidence of acclimation, but the gradual yearly suppression of these enzymes suggests the system has not reach a steady state.

Glycolysis Is Dynamic and Relates Closely to Respiration Rate in Stored Sugarbeet Roots

PubMed Central

Megguer, Clarice A.; Fugate, Karen K.; Lafta, Abbas M.; Ferrareze, Jocleita P.; Deckard, Edward L.; Campbell, Larry G.; Lulai, Edward C.; Finger, Fernando L.

2017-01-01

Although respiration is the principal cause of the loss of sucrose in postharvest sugarbeet (Beta vulgaris L.), the internal mechanisms that control root respiration rate are unknown. Available evidence, however, indicates that respiration rate is likely to be controlled by the availability of respiratory substrates, and glycolysis has a central role in generating these substrates. To determine glycolytic changes that occur in sugarbeet roots after harvest and to elucidate relationships between glycolysis and respiration, sugarbeet roots were stored for up to 60 days, during which activities of glycolytic enzymes and concentrations of glycolytic substrates, intermediates, cofactors, and products were determined. Respiration rate was also determined, and relationships between respiration rate and glycolytic enzymes and metabolites were evaluated. Glycolysis was highly variable during storage, with 10 of 14 glycolytic activities and 14 of 17 glycolytic metabolites significantly altered during storage. Changes in glycolytic enzyme activities and metabolites occurred throughout the 60 day storage period, but were greatest in the first 4 days after harvest. Positive relationships between changes in glycolytic enzyme activities and root respiration rate were abundant, with 10 of 14 enzyme activities elevated when root respiration was elevated and 9 glycolytic activities static during periods of unchanging respiration rate. Major roles for pyruvate kinase and phosphofructokinase in the regulation of postharvest sugarbeet root glycolysis were indicated based on changes in enzymatic activities and concentrations of their substrates and products. Additionally, a strong positive relationship between respiration rate and pyruvate kinase activity was found indicating that downstream TCA cycle enzymes were unlikely to regulate or restrict root respiration in a major way. Overall, these results establish that glycolysis is not static during sugarbeet root storage and that changes in glycolysis are closely related to changes in sugarbeet root respiration. PMID:28596778

A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

PubMed

Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

2010-01-01

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.

Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262.

PubMed

Nascimento, Thiago Pajeú; Sales, Amanda Emmanuelle; Porto, Tatiana Souza; Costa, Romero Marcos Pedrosa Brandão; Breydo, Leonid; Uversky, Vladimir N; Porto, Ana Lúcia Figueiredo; Converti, Attilio

2017-08-01

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu 2+ , Mg 2+ , and Fe 2+ . The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

Characterization of the glucansucrase GTF180 W1065 mutant enzymes producing polysaccharides and oligosaccharides with altered linkage composition.

PubMed

Meng, Xiangfeng; Pijning, Tjaard; Tietema, Martin; Dobruchowska, Justyna M; Yin, Huifang; Gerwig, Gerrit J; Kralj, Slavko; Dijkhuizen, Lubbert

2017-02-15

Exopolysaccharides produced by lactic acid bacteria are extensively used for food applications. Glucansucrase enzymes of lactic acid bacteria use sucrose to catalyze the synthesis of α-glucans with different linkage compositions, size and physico-chemical properties. Crystallographic studies of GTF180-ΔN show that at the acceptor binding sites +1 and +2, residue W1065 provides stacking interactions to the glucosyl moiety. However, the detailed functional roles of W1065 have not been elucidated. We performed random mutagenesis targeting residue W1065 of GTF180-ΔN, resulting in the generation of 10 mutant enzymes that were characterized regarding activity and product specificity. Characterization of mutant enzymes showed that residue W1065 is critical for the activity of GTF180-ΔN. Using sucrose, and sucrose (donor) plus maltose (acceptor) as substrates, the mutant enzymes synthesized polysaccharides and oligosaccharides with changed linkage composition. The stacking interaction of an aromatic residue at position 1065 is essential for polysaccharide synthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

PubMed

Alonso-Andrés, Patricia; Albasanz, José Luis; Ferrer, Isidro; Martín, Mairena

2018-01-24

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines. © 2018 International Society of Neuropathology.

Glucose 6-phosphate dehydrogenase and the kidney.

PubMed

Spencer, Netanya Y; Stanton, Robert C

2017-01-01

Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. G6PD is the main source of the essential cellular reductant, NADPH. The purpose of this review is to describe the biochemistry of G6PD and NADPH, cellular factors that regulate G6PD, normal physiologic roles of G6PD, and the pathogenic role altered G6PD/NADPH plays in kidney disease. NADPH is required for many essential cellular processes such as the antioxidant system, nitric oxide synthase, cytochrome p450 enzymes, and NADPH oxidase. Decreased G6PD activity and, as a result, decreased NADPH level have been associated with diabetic kidney disease, altered nitric oxide production, aldosterone-mediated endothelial dysfunction, and dialysis-associated anemia. Increased G6PD activity is associated with all cancers including kidney cancer. Inherited G6PD deficiency is the most common mutation in the world that is thought to be a relatively mild disorder primarily associated with anemia. Yet, intriguing studies have shown an increased prevalence of diabetes mellitus in G6PD-deficient people. It is not known if G6PD-deficient people are at more risk for other diseases. Much more research needs to be done to determine the role of altered G6PD activity (inherited or acquired) in the pathogenesis of kidney disease.

[Prolyl hydroxylase activity in liver specimens in chronic liver diseases (author's transl)].

PubMed

Langness, U; Clausnitzer, H; Verspohl, M; Grasedyck, K

1978-08-25

100 patients were laparoscopied, liver tissue specimens taken from atypically altered areas. Prolyl hydroxylase was determined in the specimen, in parallel tissue was examined by light microscope. 8 groups of patients could be differentiated: Patients 1. with active, 2, with inactive cirrhosis, 3. with fatty infiltrations, 4. with fatty infiltration and mesenchymal reaction, 5. with aggressive, 6. with persistent, 7. with reactive hepatitis, 8. patients without histological changes. In the case of connective tissue increase in the liver prolyl hydroxylase activities were statistically significant above normal. In addition, there was a statistically significant difference between the enzyme activities of each group. A correlation could be found between prolyl hydroxylase activity and morphologically estimated connective tissue formation, but not the serum enzyme activities usually determined in liver diseases. Therefore, could be concluded that prolyl hydroxylase activity is an index of actual collagen biosynthesis in chronic liver diseases.

Transient postnatal fluoxetine leads to decreased brain arachidonic acid metabolism and cytochrome P450 4A in adult mice.

PubMed

Ramadan, Epolia; Blanchard, Helene; Cheon, Yewon; Fox, Meredith A; Chang, Lisa; Chen, Mei; Ma, Kaizong; Rapoport, Stanley I; Basselin, Mireille

2014-05-01

Fetal and perinatal exposure to selective serotonin (5-HT) reuptake inhibitors (SSRIs) has been reported to alter childhood behavior, while transient early exposure in rodents is reported to alter their behavior and decrease brain extracellular 5-HT in adulthood. Since 5-HT2A/2C receptor-mediated neurotransmission can involve G-protein coupled activation of cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (ARA) from synaptic membrane phospholipid, we hypothesized that transient postnatal exposure to fluoxetine would alter brain ARA metabolism in adult mice. Brain ARA incorporation coefficients k* and rates Jin were quantitatively imaged following intravenous [1-(14)C]ARA infusion of unanesthetized adult mice that had been injected daily with fluoxetine (10mg/kg i.p.) or saline during postnatal days P4-P21. Expression of brain ARA metabolic enzymes and other relevant markers also was measured. On neuroimaging, k* and Jin was decreased widely in early fluoxetine- compared to saline-treated adult mice. Of the enzymes measured, cPLA2 activity was unchanged, while Ca(2+)-independent iPLA2 activity was increased. There was a significant 74% reduced protein level of cytochrome P450 (CYP) 4A, which can convert ARA to 20-HETE. Reduced brain ARA metabolism in adult mice transiently exposed to postnatal fluoxetine, and a 74% reduction in CYP4A protein, suggest long-term effects independent of drug presence in brain ARA metabolism, and in CYP4A metabolites. These changes might contribute to reported altered behavior following early SSRI in rodents. Published by Elsevier Ltd.

Zn2+, not Ca2+, is the most effective cation for activation of dolichol kinase of mammalian brain.

PubMed

Sakakihara, Y; Volpe, J J

1985-12-15

The cation specificity of dolichol kinase of mammalian brain and the potential involvement of a Ca2+-calmodulin system in regulation of this enzyme have been studied. Among 10 divalent cations examined, Zn2+ was found to be most effective for the activation of dolichol kinase of rat and calf brain and cultured C-6 glial cells. The activations with Ca2+, Co2+, and Mg2+ were 53%, 32%, and 18% of the full activation with Zn2+, respectively. No combinations of the cations could activate the enzyme as much as Zn2+ alone. A role for a Ca2+-calmodulin system in the regulation of brain dolichol kinase was not supported by our data. First, the concentration of free Ca2+ required for the maximum activation of dolichol kinase was two to three orders of magnitude greater than the concentration required by typical calmodulin-dependent enzymes. Second, neither the depletion of calmodulin from the microsomal fraction nor the addition of exogenous calmodulin caused an alteration in the activation of dolichol kinase by Ca2+ (or Zn2+). Third, antagonists of calmodulin failed to suppress the activation of the enzyme by Ca2+ (or Zn2+). The data raise the possibility that Zn2+ is involved in the regulation of dolichol kinase in brain.

The effect of enzymes upon metabolism, storage, and release of carbohydrates in normal and abnormal endometria.

PubMed

Hughes, E C

1976-07-01

This paper presents preliminary data concerning the relationship of various components of glandular epithelium and effect of enzymes on metabolism, storage, and release of certain substances in normal and abnormal endometria. Activity of these endometrial enzymes has been compared between two groups: 252 patients with normal menstrual histories and 156 patients, all over the age of 40, with abnormal uterine bleeding. Material was obtained by endometrial biopsy or curettage. In the pathologic classification of the group of 156, 30 patients had secretory endometria, 88 patients had endometria classified as proliferative, 24 were classified as endometrial hyperplasia, and 14 were classified as adenocarcinoma. All tissue was studied by histologic, histochemical, and biochemical methods. Glycogen synthetase activity caused synthesis of glucose to glycogen, increasing in amount until midcycle, when glycogen phosphorylase activity caused the breakdown to glucose during the regressive stage of endometrial activity. This normal cyclic activity did not occur in the abnormal endometria, where activity of both enzymes continued at low constant tempo. Only the I form of glycogen synthetase increased as the tissue became more hyperplastic. With the constant glycogen content and the increased activity of both the TPN isocitric dehydrogenase and glucose-6-phosphate dehydrogenase in the hyperplastic and cancerous endometria, tissue energy was created, resulting in abnormal cell proliferation. These altered biochemical and cellular activities may be the basis for malignant cell growth.

The Effects of Subacute Exposure of Peracetic Acid on Lipid Peroxidation and Hepatic Enzymes in Wistar Rats

PubMed Central

Marjani, Abdoljalal; Golalipour, Mohammad J.; Gharravi, Anneh M.

2010-01-01

Objectives This study was undertaken to determine the effect of subacute exposure of peracetic acid on lipid peroxidation and hepatic enzymes in Wistar rats. Methods 48 male animals in Treatment Group I, II and III received 0.2%, 2% and 20% peracetic acid daily for 2 and 4 weeks. Results Serum malondialdehyde increased and Alanine Transaminase and Aspartate Transaminase decreased significantly in groups 2 and 3, compared to the control group. The malondialdehyde, Alanine Transaminase and Aspartate Transaminase with 0.2% and 2% doses of peracetic acid for 2 weeks do not lead to the alteration of malondialdehyde and enzyme activities. Conclusion This study demonstrated that the enhancement of malondialdehyde could provide an oxidative damage induced by disinfectant peroxidation at 20% and 2% doses at 2 and 4 weeks. The consumption of peroxidation with 20% for 2 weeks and 2% for 4 weeks can cause the increase of malondialdehyde and the decrease of enzyme activities, respectively. PMID:22043353

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

PubMed Central

Witmer, M. R.; Palmieri-Young, D.; Villafranca, J. J.

1994-01-01

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS. PMID:7849593

Copper-induced alteration in sucrose partitioning and its relationship to the root growth of two Elsholtzia haichowensis Sun populations.

PubMed

Li, Min-Jing; Xiong, Zhi-Ting; Liu, Hui; Kuo, Yi-Ming; Tong, Lei

2016-10-02

Hydroponic culture was used to comparatively investigate the copper (Cu)-induced alteration to sucrose metabolism and biomass allocation in two Elsholtzia haichowensis Sun populations with one from a Cu-contaminated site (CS) and the other from a non-contaminated site (NCS). Experimental results revealed that biomass allocation preferred roots over shoots in CS population, and shoots over roots in NCS population under Cu exposure. The difference in biomass allocation was correlated with the difference in sucrose partitioning between the two populations. Cu treatment (45 μM) significantly decreased leaf sucrose content and increased root sucrose content in CS population as a result of the increased activities of leaf sucrose synthesis enzymes (sucrose phosphate synthetase and sucrose synthase) and root sucrose cleavage enzyme (vacuolar invertase), which led to increased sucrose transport from leaves to roots. In contrast, higher Cu treatment increased sucrose content in leaves and decreased sucrose content in roots in NCS population as a result of the decreased activities of root sucrose cleavage enzymes (vacuolar and cell wall invertases) that led to less sucrose transport from leaves to roots. These results provide important insights into carbon resource partitioning and biomass allocation strategies in metallophytes and are beneficial for the implementation of phytoremediation techniques.

Evidence for symbiont-induced alteration of a host's gene expression: irreversible loss of SAM synthetase from Amoeba proteus.

PubMed

Choi, J Y; Lee, T W; Jeon, K W; Ahn, T I

1997-01-01

Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X-bacteria supply the needed enzyme. A monoclonal antibody against the 45-kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase activity. These results show how symbiotic X-bacteria may become essential cellular components of amoeba by supplementing a genetic defect for an amoeba's house-keeping gene that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Protective effects of Sonchus asper against KBrO3 induced lipid peroxidation in rats

PubMed Central

2012-01-01

Background Sonchus asper is traditionally used in Pakistan for the treatment of reproductive dysfunction and oxidative stress. The present investigation was aimed to evaluate chloroform extract of Sonchus asper (SACE) against potassium bromate-induced reproductive stress in male rats. Methods 20 mg/kg body weight (b.w.) potassium bromate (KBrO3) was induced in 36 rats for four weeks and checked the protective efficacy of SACE at various hormonal imbalances, alteration of antioxidant enzymes, and DNA fragmentation levels. High performance chromatography (HPLC) was used for determination of bioactive constituents responsible. Results The level of hormonal secretion was significantly altered by potassium bromate. DNA fragmentation%, activity of antioxidant enzymes; catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and phase II metabolizing enzymes viz; glutathione reductase (GSR), glutathione peroxidase (GSHpx), glutathione-S-tansase (GST) and reduced glutathione (GSH) was decreased while hydrogen per oxide contents and thiobarbituric acid reactive substances (TBARS) were increased with KBrO3 treatment. Treatment with SACE effectively ameliorated the alterations in the biochemical markers; hormonal and molecular levels while HPLC characterization revealed the presence of catechin, kaempferol, rutin and quercetin. Conclusion Protective effects of Sonchus asper vs. KBrO3 induced lipid peroxidation might be due to bioactive compound present in SACE. PMID:23186106

Protective effects of Sonchus asper against KBrO3 induced lipid peroxidation in rats.

PubMed

Khan, Rahmat Ali; Khan, Muhammad Rashid; Sahreen, Sumaira

2012-11-27

Sonchus asper is traditionally used in Pakistan for the treatment of reproductive dysfunction and oxidative stress. The present investigation was aimed to evaluate chloroform extract of Sonchus asper (SACE) against potassium bromate-induced reproductive stress in male rats. 20 mg/kg body weight (b.w.) potassium bromate (KBrO3) was induced in 36 rats for four weeks and checked the protective efficacy of SACE at various hormonal imbalances, alteration of antioxidant enzymes, and DNA fragmentation levels. High performance chromatography (HPLC) was used for determination of bioactive constituents responsible. The level of hormonal secretion was significantly altered by potassium bromate. DNA fragmentation%, activity of antioxidant enzymes; catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and phase II metabolizing enzymes viz; glutathione reductase (GSR), glutathione peroxidase (GSHpx), glutathione-S-tansase (GST) and reduced glutathione (GSH) was decreased while hydrogen per oxide contents and thiobarbituric acid reactive substances (TBARS) were increased with KBrO3 treatment. Treatment with SACE effectively ameliorated the alterations in the biochemical markers; hormonal and molecular levels while HPLC characterization revealed the presence of catechin, kaempferol, rutin and quercetin. Protective effects of Sonchus asper vs. KBrO3 induced lipid peroxidation might be due to bioactive compound present in SACE.

Time-measurement-regulating peptide PIN may alter a timer conformation of Time Interval Measuring Enzyme (TIME).

PubMed

Ti, Xiaonan; Tani, Naoki; Isobe, Minoru; Kai, Hidenori

2006-05-01

The TIME (Time Interval Measuring Enzyme) ATPase measures time intervals in accordance with diapause development, which indispensably requires cold for resumption of embryonic development in the silkworm (Bombyx mori). The PIN (Peptidyl Inhibitory Needle) peptide regulates the time measurement function of TIME. In the present study we investigated the interaction between TIME and PIN in order to address the mechanism of diapause development. When TIME was isolated from eggs later than 12 days after oviposition, transient bursts of ATPase activity occurred 18h after isolation of TIME, and the younger the eggs and pupal ovaries from which TIME was isolated, the earlier the bursts of ATPase activity appeared. However, no interval-timer activation of ATPase occurred in ovaries earlier than 6 days after pupation. Similar patterns of ATPase activity occurred in test tubes after mixing TIME with PIN. The shorter the time PIN was mixed with TIME, the earlier the ATPase activity appeared. The timer may be built into the protein conformation of TIME, and PIN (which is present in ovaries beginning 6 days after pupation) appears able to alter this timer conformation through pupal stages to laid eggs. We discuss the possible mechanism of diapause development in relation to the timer mechanism of TIME.

Steroid Hormones and Uterine Vascular Adaptation to Pregnancy

PubMed Central

Chang, Katherine; Zhang, Lubo

2008-01-01

Pregnancy is a physiological state that involves a significant decrease in uterine vascular tone and an increase in uterine blood flow, which is mediated in part by steroid hormones, including estrogen, progesterone, and cortisol. Previous studies have demonstrated the involvement of these hormones in the regulation of uterine artery contractility through signaling pathways specific to the endothelium and the vascular smooth muscle. Alterations in endothelial nitric oxide synthase expression and activity, nitric oxide production, and expression of enzymes involved in PGI2 production contribute to the uterine artery endothelium-specific responses. Steroid hormones also have an effect on calcium-activated potassium channel activity, PKC signaling pathway and myogenic tone, and alterations in pharmacomechanical coupling in the uterine artery smooth muscle. This review addresses current understanding of the molecular mechanisms by which steroid hormones including estrogen, progesterone, and cortisol modulate uterine artery contractility to alter uterine blood flow during pregnancy with an emphasis on the pregnant ewe model. PMID:18497342

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

NASA Technical Reports Server (NTRS)

Roux, S. J.

1990-01-01

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Altered precipitation regime affects the function and composition of soil microbial communities on multiple time scales.

PubMed

Zeglin, L H; Bottomley, P J; Jumpponen, A; Rice, C W; Arango, M; Lindsley, A; McGowan, A; Mfombep, P; Myrold, D D

2013-10-01

Climate change models predict that future precipitation patterns will entail lower-frequency but larger rainfall events, increasing the duration of dry soil conditions. Resulting shifts in microbial C cycling activity could affect soil C storage. Further, microbial response to rainfall events may be constrained by the physiological or nutrient limitation stress of extended drought periods; thus seasonal or multiannual precipitation regimes may influence microbial activity following soil wet-up. We quantified rainfall-driven dynamics of microbial processes that affect soil C loss and retention, and microbial community composition, in soils from a long-term (14-year) field experiment contrasting "Ambient" and "Altered" (extended intervals between rainfalls) precipitation regimes. We collected soil before, the day following, and five days following 2.5-cm rainfall events during both moist and dry periods (June and September 2011; soil water potential = -0.01 and -0.83 MPa, respectively), and measured microbial respiration, microbial biomass, organic matter decomposition potential (extracellular enzyme activities), and microbial community composition (phospholipid fatty acids). The equivalent rainfall events caused equivalent microbial respiration responses in both treatments. In contrast, microbial biomass was higher and increased after rainfall in the Altered treatment soils only, thus microbial C use efficiency (CUE) was higher in Altered than Ambient treatments (0.70 +/- 0.03 > 0.46 +/- 0.10). CUE was also higher in dry (September) soils. C-acquiring enzyme activities (beta-glucosidase, cellobiohydrolase, and phenol oxidase) increased after rainfall in moist (June), but not dry (September) soils. Both microbial biomass C:N ratios and fungal:bacterial ratios were higher at lower soil water contents, suggesting a functional and/or population-level shift in the microbiota at low soil water contents, and microbial community composition also differed following wet-up and between seasons and treatments. Overall, microbial activity may directly (C respiration) and indirectly (enzyme potential) reduce soil organic matter pools less in drier soils, and soil C sequestration potential (CUE) may be higher in soils with a history of extended dry periods between rainfall events. The implications include that soil C loss may be reduced or compensated for via different mechanisms at varying time scales, and that microbial taxa with better stress tolerance or growth efficiency may be associated with these functional shifts.

Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress.

PubMed

Singh, Raja B; Hryshko, Larry; Freed, Darren; Dhalla, Naranjan S

2012-02-01

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

Nitrogen inputs accelerate phosphorus cycling rates across a wide variety of terrestrial ecosystems.

PubMed

Marklein, Alison R; Houlton, Benjamin Z

2012-02-01

• Biologically essential elements--especially nitrogen (N) and phosphorus (P)--constrain plant growth and microbial functioning; however, human activities are drastically altering the magnitude and pattern of such nutrient limitations on land. Here we examine interactions between N and P cycles of P mineralizing enzyme activities (phosphatase enzymes) across a wide variety of terrestrial biomes. • We synthesized results from 34 separate studies and used meta-analysis to evaluate phosphatase activity with N, P, or N×P fertilization. • Our results show that N fertilization enhances phosphatase activity, from the tropics to the extra-tropics, both on plant roots and in bulk soils. By contrast, P fertilization strongly suppresses rates of phosphatase activity. • These results imply that phosphatase enzymes are strongly responsive to changes in local nutrient cycle conditions. We also show that plant phosphatases respond more strongly to fertilization than soil phosphatases. The tight coupling between N and P provides a mechanism for recent observations of N and P co-limitation on land. Moreover, our results suggest that terrestrial plants and microbes can allocate excess N to phosphatase enzymes, thus delaying the onset of single P limitation to plant productivity as can occur via human modifications to the global N cycle. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells.

PubMed

Jastreboff, M M; Kedzierska, B; Rode, W

1983-07-15

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia.

PubMed

Timson, David J; Lindert, Steffen

2013-09-10

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. Copyright © 2013 Elsevier B.V. All rights reserved.

Elongation factor Tu resistant to kirromycin in an Esherichia coli mutant altered in both tuf genes

PubMed Central

Fischer, Eckhard; Wolf, Heinz; Hantke, Klaus; Parmeggiani, Andrea

1977-01-01

A mutant of Escherichia coli is described that displays kirromycin resistance in a cell-free system by virtue of an altered elongation factor Tu (EF-Tu). In poly(U)-directed poly(Phe) synthesis the kirromycin resistance of the crystallized enzyme ranged between a factor of 80 and 700, depending on temperature. Similarly, kirromycin-induced EF-Tu GTPase activity uncoupled from ribosomes and aminoacyl-tRNA required correspondingly higher concentrations of the antibiotic. Resistance of EF-Tu to kirromycin is a consequence of a modified enzyme structure as indicated by its altered fingerprint pattern. P1 transduction experiments showed that the kirromycin-resistant EF-Tu is coded by an altered tufB gene (tufB1). The known existence of two genes coding for EF-Tu would interfere with the recognition of a mutant altered in only one of those genes, if the mutation were recessive. Because kirromycin blocks EF-Tu release from the ribosome, kirromycin sensitivity is dominant, as shown by the failure of a mixed EF-Tu population to express resistance in vitro. Therefore, phenotypic expression of kirromycin resistance in vivo appears to be only possible if the EF-Tu mutant lacks an active tufA gene, a property likely to be inherited from the parental D22 strain. The observations that introduction of a tufA+ region makes the resistant strain sensitive to the antibiotic and that transduction of tufB1 into a recipient other than E. coli D22 yields kirromycin-sensitive progeny support these conclusions. Images PMID:337296

Variant amino acid residues alter the enzyme activity of peanut type 2 Diacylglycerol Acyltransferases

USDA-ARS?s Scientific Manuscript database

Diacylglycerol acyltransferase (DGAT) catalyzes the final, rate-limiting step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. In this study, type-2 DGAT2 genes were cloned from eleven peanut cultivars. Sequence analysis revealed at least eight peanut D...

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase

USDA-ARS?s Scientific Manuscript database

Nucleotide-activated sugars are essential substrates for plant cell wall carbohydrate-polymer biosynthetic glycosyltransferase enzymes. The most prevalent sugars in grass cell walls include glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the uridine di...

Ovine maternal nutrient restriction from mid to late gestation decreases heptic progesterone inactivating enzyme activity

USDA-ARS?s Scientific Manuscript database

Previously we have shown increased concentrations of progesterone and decreased liver weight in mid to late pregnant ewes provided a nutrient restricted vs. adequate diet. This alteration in peripheral progesterone could be due to increased synthesis and/or decreased clearance of progesterone. There...

Effects of sulfate deposition on pore water dissolved organic carbon, nutrients, and microbial enzyme activities in a northern peatland

EPA Science Inventory

Export of dissolved organic carbon from lakes and streams has increased throughout Europe and North America over the past several decades. One possible cause is altered deposition chemistry; specifically, decreasing sulfate inputs leading to changes in ionic strength and dissolve...

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

PubMed Central

2013-01-01

Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement. PMID:23819621

Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function.

PubMed

Sunitha, Balaraju; Gayathri, Narayanappa; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Nalini, Atchayaram; Padmanabhan, Balasundaram; Srinivas Bharath, Muchukunte Mukunda

2016-07-01

Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from dysferlinopathy (Dysfy), polymyositis (PM), and distal myopathy with rimmed vacuoles (DMRV) displayed morphological and biochemical evidences of mitochondrial dysfunction. Proteomic analysis revealed down-regulation of electron transport chain (ETC) subunits, assembly factors, and tricarboxylic acid (TCA) cycle enzymes, with 80 proteins common among the three pathologies. Mitochondrial proteins from muscle pathologies also displayed higher Trp oxidation that could alter the local structure. Cover image for this issue: doi: 10.1111/jnc.13324. © 2016 International Society for Neurochemistry.

Assessment of environmental factors affecting male fertility

PubMed Central

Dixon, R. L.; Sherins, R. J.; Lee, I. P.

1979-01-01

Exposure to drinking water containing as much as 500 ppm aluminum chloride for periods of 30, 60, and 90 days had no apparent effect on male reproductive processes. In an attempt to correlate enzyme activity with particular spermatogenic cell types, postnatal development of testicular enzymes was studied. Eight enzymes were selected: hyaluronidase (H), lactate dehydrogenase isoenzyme-X (LDH-X), dehydrogenases of sorbitol (SDH), α-glycerophosphate (GPDH), glucose-6-phosphate (G6PDH), malate (MDH), glyceraldehyde-3-phosphate (G3PDH), and isocitrate (ICDH). Enzyme specific activities in testicular homogenates were determined. Two types of enzyme developmental patterns were observed. One was represented by H, LDH-X, SDH, and GPDH; and the other by G6PDH, MDH, G3PDH, and ICDH. The former was characterized by a change in enzyme activities from low in newborn to high in adult while in the latter this pattern was reversed. The two complementary enzyme systems crossed each other at puberty. Prior to puberty, only spermatogonial cells are present; sperm differentiation initiated at puberty adds spermatocytes and spermatids to the testicular cell population. Male rats were exposed to borax in their diet for periods of 30 and 60 days. Concentrations of boron were 0, 500, 1000, and 2000 ppm. At the end of each experimental period, the specific activities of the selected enzymes were determined in the testis and prostate. Correlations of enzyme activity with testicular histology and androgen activities of the male accessory organs were sought. In addition, plasma FSH, LH, and testosterone levels were measured to assess pituitary-testicular interaction. Plasma and testicular boron concentrations were determined and a minimum boron concentration which induced germinal aplasia and male infertility was estimated. In both 30 and 60 day feeding studies, male rats receiving 500 ppm failed to demonstrate any significant adverse effects. In contrast, male rats receiving 100 and 2000 ppm boron displayed a significant loss of germinal elements, although most of the Leydig and Sertoli cells appeared normal. Testicular atrophy was associated with a decrease in seminiferous tubular diameter and a marked reduction of spermatocytes and spermatogenic cells. These morphologic alterations were associated with a concomitant reduction of H, SDH, and LDH-X specific activities. In contrast, the specific activities of G3PDH and MDH were significantly elevated above control. The increase in these enzyme activities can be attributed to the relative enrichment of spermatogonial cells during the loss of spermatocytes and spermiogenic cells. Boron-induced male germinal aplasia was also associated with significantly elevated plasma FSH while plasma LH and testosterone levels were not significantly altered. Plasma testosterone levels were unaltered. Male fertility studies demonstrated that at the 500 ppm boron level, fertility was unaffected. However, at 1000 and 2000 ppm boron, male fertility was significantly reduced. Most effects were reversible within 5 weeks. However, the male group receiving 2000 ppm boron for 60 days remained sterile. There was no dose-related decrease in litter size or fetal death in utero. Therefore, the boron-induced infertility was apparently not due to a dominant lethal effect but rather to germinal aplasia. Boron appears toxic to spermatogenic cells at testicular concentrations of 6–8 ppm. ImagesFIGURE 6.FIGURE 9. PMID:446458

Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer

PubMed Central

Sen, Prakriti; Ganguly, Pooja; Ganguly, Niladri

2018-01-01

Human papillomaviruses (HPVs) are double stranded circular DNA viruses that infect cutaneous and mucosal epithelial cells. Almost 99% of cervical cancer has a HPV infection. The early oncoproteins E6 and E7 are important in this cellular transformation process. Epigenetic mechanisms have long been known to result in decisive alterations in DNA, leading to alterations in DNA-protein interactions, alterations in chromatin structure and compaction and significant alterations in gene expression. The enzymes responsible for these epigenetic modifications are DNA methyl transferases (DNMTs), histone acetylases and deacetylases. Epigenetics has an important role in cancer development by modifying the cellular micro environment. In this review, the authors discuss the role of HPV oncoproteins E6 and E7 in modulating the epigenetic mechanisms inside the host cell. The oncoproteins induce the expression of DNMTs which lead to aberrant DNA methylations and disruption of the normal epigenetic processes. The E7 oncoprotein may additionally directly bind and induce methyl transferase activity of the enzyme. These modulations lead to altered gene expression levels, particularly the genes involved in apoptosis, cell cycle and cell adhesion. In addition, the present review discusses how epigenetic mechanisms may be targeted for possible therapeutic interventions for HPV mediated cervical cancer. PMID:29285184

Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

PubMed

Timms, Nicole; Windle, Claire L; Polyakova, Anna; Ault, James R; Trinh, Chi H; Pearson, Arwen R; Nelson, Adam; Berry, Alan

2013-03-04

Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

Thyroid hormone stimulates myoglobin expression in soleus and extensorum digitalis longus muscles of rats: concomitant alterations in the activities of Krebs cycle oxidative enzymes.

PubMed

dos Santos, R A; Giannocco, G; Nunes, M T

2001-06-01

Myoglobin (Mb) gene expression, Citrate Synthase (CS) and Succinate Dehydrogenase (SDH) activities of Soleus (S) and Extensorum Digitalis Longus (EDL) muscles were studied in intact, thyroidectomized and T3-treated (25 microg/100g, BW, ip, 15 days) rats. The fiber type composition of S muscle was also evaluated and used as control of the T3-induced effects. In the S muscle, the T3 treatment increased the Mb mRNA and protein expression, as well as the CS and SDH activity. These changes occurred parallel to the expected increase in type II (fast) and decrease in type I (slow)-fibers in S muscle. In the hypothyroid state, the Mb mRNA was decreased, while the Mb expression and CS activity tended to decrease. In contrast the SDH activity was increased, probably due to the enhanced motor activity that occurs as a short-term response to the hypothermia induced by hypothyroidism. In the EDL, the alterations were milder than those in S muscle in both thyroid states. These findings show that Mb gene expression is induced by T3. This is concomitant with the enhancement of Krebs Cycle enzyme activities and provides additional evidence that thyroid hormone increases the aerobic potential of skeletal muscles, as well as the speed of muscle contraction.

Altered Expression of Urea Cycle Enzymes in Amyloid-β Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

PubMed

Jęśko, Henryk; Lukiw, Walter J; Wilkaniec, Anna; Cieślik, Magdalena; Gąssowska-Dobrowolska, Magdalena; Murawska, Emilia; Hilgier, Wojciech; Adamczyk, Agata

2018-01-01

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

The Effects of Peatland Plant Functional Types and Altered Hydrology on Porewater Chemistry in a Northern Bog

NASA Astrophysics Data System (ADS)

Daniels, A.; Kane, E. S.; Lilleskov, E. A.; Kolka, R. K.; Chimner, R. A.; Potvin, L. R.; Romanowicz, K. J.

2012-12-01

Northern wetlands, peatlands in particular, have been shown to store around 30% of the world's soil carbon and thus play a significant role in the carbon cycle of our planet. Carbon accumulation in peatlands is the result of retarded decomposition due to low oxygen availability in these water-logged environments. Changes in our planet's climate cycles are altering peatland hydrology and vegetation communities, resulting in changes in their ability to sequester carbon through increases in peat carbon oxidation and mineralization. To date, the consequences of altered hydrology and changes in vegetation communities, and their interactive effects on carbon storage, are not well understood. We have initiated a research plan that assesses the varying roles that water table variation and vegetation communities have on extracellular enzyme activity and labile carbon availability in porewater from an ombrotrophic bog. We assessed the effects of plant functional group (ericaceous shrubs, sedges, and bryophytes) and water table position on biogeochemical processes. Specifically, we measured dissolved organic carbon (DOC), total dissolved nitrogen (TDN), enzyme activity, organic acids, anions and cations, spectral indexes of aromaticity, and phenolic content in addressing our hypotheses of responses to climate change drivers. Research on these components will evaluate the relative importance of biology, water table, and their interactive affects on the porewater quality of peatlands. We hypothesized that oxygen availability will strongly influence decomposition in these systems but that this response will largely be mediated by changes in plant community and the enzymes associated with root exudates and mycorrhizae. To date, our data confirm vegetation and water table related patterns. Acetate and propionate concentrations in the sedge-dominated communities dropped significantly with depth and drainage, relative to the control and ericaceous treatments, which likely reflects changes in redox potential owing to physiological differences in sedges which contain aerenchyma cell, and a reduction in the products of anaerobic metabolism. DOC increased in the lowered water table treatments in all vegetation community types. Enzymatic activities have changed in response to water table level and vegetation community. While we have not detected significant levels of peroxidase enzymes in porewater, initial results indicate that hydrolase enzyme activities were higher in the sedge-dominated communities with a lowered water table. Through this research, we are hoping to advance our knowledge of the drivers behind peatland biogeochemistry and how ombrotrophic peat systems may respond to climate change influences.

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide.

PubMed

Rakhmetov, A D; Pil, Lee Sang; Ostapchenko, L I; Zoon, Chae Ho

2015-01-01

Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.

In Healthy Young Men, a Short Exhaustive Exercise Alters the Oxidative Stress Only Slightly, Independent of the Actual Fitness.

PubMed

Finkler, Maya; Hochman, Ayala; Pinchuk, Ilya; Lichtenberg, Dov

2016-01-01

The aim of the present study was to evaluate the apparent disagreement regarding the effect of a typical cycling progressive exercise, commonly used to assess VO2max, on the kinetics of ex vivo copper induced peroxidation of serum lipids. Thirty-two (32) healthy young men, aged 24-30 years, who do not smoke and do not take any food supplements, participated in the study. Blood was withdrawn from each participant at three time points (before the exercise and 5 minutes and one hour after exercise). Copper induced peroxidation of sera made of the blood samples was monitored by spectrophotometry. For comparison, we also assayed TBARS concentration and the activity of oxidation-related enzymes. The physical exercise resulted in a slight and reversible increase of TBARS and slight changes in the activities of the studied antioxidant enzymes and the lag preceding peroxidation did not change substantially. Most altered parameters returned to baseline level one hour after exercise. Notably, the exercise-induced changes in OS did not correlate with the physical fitness of the subjects, as evaluated in this study (VO2max = 30-60 mL/min/kg). We conclude that in healthy young fit men a short exhaustive exercise alters only slightly the OS, independent of the actual physical fitness.

Alteration in metabolism and toxicity of acetaminophen upon repeated administration in rats.

PubMed

Kim, Sun J; Lee, Min Y; Kwon, Do Y; Kim, Sung Y; Kim, Young C

2009-10-01

Our previous studies showed that administration of a subtoxic dose of acetaminophen (APAP) to female rats increased generation of carbon monoxide from dichloromethane, a metabolic reaction catalyzed mainly by cytochrome P450 (CYP) 2E1. In this study we examined the changes in metabolism and toxicity of APAP upon repeated administration. An intraperitoneal dose of APAP (500 mg/kg) alone did not increase aspartate aminotransferase, alanine aminotransferase, or sorbitol dehydrogenase activity in serum, but was significantly hepatotoxic when the rats had been pretreated with an identical dose of APAP 18 h earlier. The concentrations and disappearance of APAP and its metabolites in plasma were monitored for 8 h after the treatment. APAP pretreatment reduced the elevation of APAP-sulfate, but increased APAP-cysteine concentrations in plasma. APAP or APAP-glucuronide concentrations were not altered. Administration of a single dose of APAP 18 h before sacrifice increased microsomal CYP activities measured with p-nitrophenol, p-nitroanisole, and aminopyrine as probes. Expression of CYP2E1, CYP3A, and CYP1A proteins in the liver was also elevated significantly. The results suggest that administration of APAP at a subtoxic dose may result in an induction of hepatic CYP enzymes, thereby altering metabolism and toxicological consequences of various chemical substances that are substrates for the same enzyme system.

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation.

PubMed

Griffeth, L K; Rosen, G M; Rauckman, E J

1985-01-01

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.

Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

PubMed

Narayan, Shoba; Devi, R S; Devi, C S Shyamala

2007-11-20

Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

PubMed

Díaz, M E; Rocha, G F; Kise, F; Rosso, A M; Guevara, M G; Parisi, M G

2018-05-08

Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l -1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l -1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would represent a new alternative to avoid the problems of environmental pollution and antimicrobial resistance. © 2018 The Society for Applied Microbiology.

F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations

PubMed Central

Mancini, Giordano; Zazza, Costantino

2015-01-01

The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization. PMID:26415031

F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations.

PubMed

Mancini, Giordano; Zazza, Costantino

2015-01-01

The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

Ameliorative potential of gingerol: Promising modulation of inflammatory factors and lipid marker enzymes expressions in HFD induced obesity in rats.

PubMed

Brahma Naidu, Parim; Uddandrao, V V Sathibabu; Ravindar Naik, Ramavat; Suresh, Pothani; Meriga, Balaji; Begum, Mustapha Shabana; Pandiyan, Rajesh; Saravanan, Ganapathy

2016-01-05

Obesity, generally linked to hyperlipidemia, has been occurring of late with distressing alarm and has now become a global phenomenon casting a huge economic burden on the health care system of countries around the world. The present study investigated the effects of gingerol over 30 days on the changes in HFD-induced obese rats in marker enzymes of lipid metabolism such as fatty-acid synthase (FAS), Acetyl CoA Carboxylase (ACC), Carnitine Palmitoyl Transferase-1(CPT-1), HMG co-A Reductase (HMGR), Lecithin Choline Acyl Transferase (LCAT) and Lipoprotein Lipase (LPL) and inflammatory markers (TNF-α and IL-6). The rats were treated orally with gingerol (75 mg kg(-1)) once daily for 30 days with a lorcaserin-treated group (10 mg kg(-1)) included for comparison. Changes in body weight, glucose, insulin resistance and expressions of lipid marker enzymes and inflammatory markers in tissues were observed in experimental rats. The administration of gingerol resulted in a significant reduction in body weight gain, glucose and insulin levels, and insulin resistance, which altered the activity, expressions of lipid marker enzymes and inflammatory markers. It showed that gingerol had significantly altered these parameters when compared with HFD control rats. This study confirms that gingerol prevents HFD-induced hyperlipidemia by modulating the expression of enzymes important to cholesterol metabolism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effect of single and binary combinations of plant-derived molluscicides on different enzyme activities in the nervous tissue of Achatina fulica.

PubMed

Rao, I G; Singh, Amrita; Singh, V K; Singh, D K

2003-01-01

Effect of single and binary treatments of plant-derived molluscicides on different enzymes--acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and acid/alkaline phosphatase (ACP/ALP)--in the nervous tissue of the harmful terrestrial snail Achatina fulica were studied. Sublethal in vivo 24-h exposure to 40% and 80% LC(50) of Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, Nerium indicum bark powder and binary combinations of A. sativum (AS) + C. deodara (CD) and CD + A. indica (AI) oils significantly altered the activity of these enzymes in the nervous tissue of Achatina fulica. The binary treatment of AS + CD was more effective against AChE, LDH, and ALP than the single ones. However, binary treatment of AI + CD was more effective against ALP. Copyright 2003 John Wiley & Sons, Ltd.

Influence of altered gravity on brain cellular energy and plasma membrane metabolism of developing lower aquatic vertebrates

NASA Astrophysics Data System (ADS)

Slenzka, K.; Appel, R.; Kappel, Th.; Rahmann, H.

Biochemical analyses of the brain of cichlid fish larvae, exposed for 7 days to increased acceleration of 3g (hyper-g), revealed an increase in energy availability (succinate dehydrogenase activity, SDH), and in mitochondrial energy transformation (creatine kinase, Mi_a-CK), but no changes in an energy consumptive process (high-affinity Ca^2+-ATPase). Brain glucose-6-phosphate dehydrogenase (G6PDH) of developing fish was previously found to be increased after hyper-g exposure. Three respectively 5 hours thereafter dramatic fluctuations in enzyme activity were registered. Analysing the cytosolic or plasma membrane-located brain creatine kinase (BB-CK) of clawed toad larvae after long-term hyper-g exposure a significant increase in enzyme activity was demonstrated, whereas the activity of a high affinity Ca^2+-ATPase remained unaffected.

Variation of human intestinal gamma-glutamyl transpeptidase in ontogenetic development.

PubMed

Sobiech, K A; Szewczuk, A

1977-01-01

Activity of gamma-glutamyl transpeptidase in human intestines was measured against alpha-naphthylamide and 12 gamma-glutamyl amino acids and peptides as substrate. Distinctly altered activity was found to accompany ontogenetic development. The ratio of the transpeptidase activity tested against monoglutamyl substrates in the intestines of 7-month fetuses, newborns and adults was 15:1:4, whereas the ratio of gamma-glutamyl cyclotransferase activities in the same age groups was 1-0:1-2:1-6. Distinct differences were found in resistance to heating, sensitivity to L-serine plus borate, and other effectors, and electrophoretic mobility, between fetal gamma-glutamyl transpeptidase and the enzyme from adults, which supports the hypothesis of existence of two forms of the enzyme in the human intestines. The results suggest involvement of gamma-glutamyl transpeptidase in the pathomechanism of celiakia in children.

Influence of altered precipitation pattern on greenhouse gas emissions and soil enzyme activities in Pannonian soils

NASA Astrophysics Data System (ADS)

Forstner, Stefan Johannes; Michel, Kerstin; Berthold, Helene; Baumgarten, Andreas; Wanek, Wolfgang; Zechmeister-Boltenstern, Sophie; Kitzler, Barbara

2013-04-01

Precipitation patterns are likely to be altered due to climate change. Recent models predict a reduction of mean precipitation during summer accompanied by a change in short-term precipitation variability for central Europe. Correspondingly, the risk for summer drought is likely to increase. This may especially be valid for regions which already have the potential for rare, but strong precipitation events like eastern Austria. Given that these projections hold true, soils in this area will receive water irregularly in few, heavy rainfall events and be subjected to long-lasting dry periods in between. This pattern of drying/rewetting can alter soil greenhouse gas fluxes, creating a potential feedback mechanism for climate change. Microorganisms are the key players in most soil carbon (C) and nitrogen (N) transformation processes including greenhouse gas exchange. A conceptual model proposed by Schimel and colleagues (2007) links microbial stress-response physiology to ecosystem-scale biogeochemical processes: In order to cope with decreasing soil water potential, microbes modify resource allocation patterns from growth to survival. However, it remains unclear how microbial resource acquisition via extracellular enzymes and microbial-controlled greenhouse gas fluxes respond to water stress induced by soil drying/rewetting. We designed a laboratory experiment to test for effects of multiple drying/rewetting cycles on soil greenhouse gas fluxes (CO2, CH4, N2O, NO), microbial biomass and extracellular enzyme activity. Three soils representing the main soil types of eastern Austria were collected in June 2012 at the Lysimeter Research Station of the Austrian Agency for Health and Food Safety (AGES) in Vienna. Soils were sieved to 2mm, filled in steel cylinders and equilibrated for one week at 50% water holding capacity (WHC) for each soil. Then soils were separated into two groups: One group received water several times per week (C=control), the other group received water only once in two weeks (D=dry). Both groups received same water totals for each soil. At the end of each two week drying period, greenhouse gas fluxes were measured via an open-chamber-system (CO2, NO) and a closed-chamber-approach (CH4, N2O, CO2). Additional cylinders were harvested destructively to quantify inorganic N forms, microbial biomass C, N and extracellular enzyme activity (Cellulase, Xylanase, Protease, Phenoloxidase, Peroxidase). We hypothesize that after rewetting (1) rates of greenhouse gas fluxes will generally increase, as well as (2) extracellular enzyme activity indicating enhanced microbial activity. However, response may be different for gases and enzymes involved in the C and N cycle, respectively, as drying/rewetting stress may uncouple microbial mediated biogeochemical cycles. Results will be presented at the EGU General Assembly. Reference: Schimel, J., Balser, T.C., and Wallenstein, M. (2007). Microbial stress-response physiology and its implications for ecosystem function. Ecology 88, 1386-1394.

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

PubMed Central

Baker, D. P.; Fetler, L.; Vachette, P.; Kantrowitz, E. R.

1996-01-01

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used. PMID:8931146

Imidazole-containing phthalazine derivatives inhibit Fe-SOD performance in Leishmania species and are active in vitro against visceral and mucosal leishmaniasis.

PubMed

Sánchez-Moreno, M; Gómez-Contreras, F; Navarro, P; Marín, C; Ramírez-Macías, I; Rosales, M J; Campayo, L; Cano, C; Sanz, A M; Yunta, M J R

2015-07-01

The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1-4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

Effect of LED photobiomodulation on fluorescent light induced changes in cellular ATPases and Cytochrome c oxidase activity in Wistar rat.

PubMed

A, Ahamed Basha; C, Mathangi D; R, Shyamala

2016-12-01

Fluorescent light exposure at night alters cellular enzyme activities resulting in health defects. Studies have demonstrated that light emitting diode photobiomodulation enhances cellular enzyme activities. The objectives of this study are to evaluate the effects of fluorescent light induced changes in cellular enzymes and to assess the protective role of pre exposure to 670 nm LED in rat model. Male Wistar albino rats were divided into 10 groups of 6 animals each based on duration of exposure (1, 15, and 30 days) and exposure regimen (cage control, exposure to fluorescent light [1800 lx], LED preexposure followed by fluorescent light exposure and only LED exposure). Na + -K + ATPase, Ca 2+ ATPase, and cytochrome c oxidase of the brain, heart, kidney, liver, and skeletal muscle were assayed. Animals of the fluorescent light exposure group showed a significant reduction in Na + -K + ATPase and Ca 2+ ATPase activities in 1 and 15 days and their increase in animals of 30-day group in most of the regions studied. Cytochrome c oxidase showed increase in their level at all the time points assessed in most of the tissues. LED light preexposure showed a significant enhancement in the degree of increase in the enzyme activities in almost all the tissues and at all the time points assessed. This study demonstrates the protective effect of 670 nm LED pre exposure on cellular enzymes against fluorescent light induced change.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

PubMed Central

Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

2012-01-01

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

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

Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.

2012-08-26

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface,more » a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.« less

Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C

1979-05-01

Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The enhanced biological activity of the analog, therefore, may be due to its resistance to inactivation by enzymes on the pituitary cell surface. The membrane-associated inactivating enzyme could play an important role in vivo in determining the concentration of intact LHRH available at the receptor site which initiates gonadotropin release.

Analysis of galactosemia-linked mutations of GALT enzyme using a computational biology approach.

PubMed

Facchiano, A; Marabotti, A

2010-02-01

We describe the prediction of the structural and functional effects of mutations on the enzyme galactose-1-phosphate uridyltransferase related to the genetic disease galactosemia, using a fully computational approach. One hundred and seven single-point mutants were simulated starting from the structural model of the enzyme obtained by homology modeling methods. Several bioinformatics programs were then applied to each resulting mutant protein to analyze the effect of the mutations. The mutations have a direct effect on the active site, or on the dimer assembly and stability, or on the monomer stability. We describe how mutations may exert their effect at a molecular level by altering H-bonds, salt bridges, secondary structure or surface features. The alteration of protein stability, at level of monomer and/or dimer, is the main effect observed. We found an agreement between our results and the functional experimental data available in literature for some mutants. The data and analyses for all the mutants are fully available in the web-accessible database hosted at http://bioinformatica.isa.cnr.it/GALT.

Antioxidant and anti-inflammatory effects of virgin coconut oil supplementation abrogate acute chemotherapy oxidative nephrotoxicity induced by anticancer drug methotrexate in rats.

PubMed

Famurewa, Ademola C; Aja, Patrick M; Maduagwuna, Ekenechukwu K; Ekeleme-Egedigwe, Chima A; Ufebe, Odomero G; Azubuike-Osu, Sharon O

2017-12-01

Methotrexate (MTX) is an efficacious anticancer agent constrained in clinical use due to its toxicity on non-targeted tissue, a considerable source of worry to clinicians. Because the toxicity is associated with oxidative stress and inflammation, the study explored antioxidant and anti-inflammatory effect of virgin coconut oil (VCO) supplementation in nephrotoxicity induced by MTX in rats. Rats were randomized into 4 groups (n=6) as follows: Control group; MTX group injected with single dose of MTX (20mg/kg, ip) on day 14; VCO (5%)+MTX and VCO (15%)+MTX groups were pre-treated with VCO diet and injected with single dose of MTX (20mg/kg, ip) on day 14. After 3 days of MTX injection, serum kidney markers, renal activities of antioxidant enzymes and glutathione (GSH) content were determined. Lipid peroxidation level and inflammatory markers- interleukin-6 (IL-6), nitric oxide (NO) and C-reactive protein (CRP) were estimated in kidney. Histopathological alterations were examined for kidney damage. MTX nephrotoxicity was evidenced by markedly elevated serum renal markers along with significant decreases in renal GSH and activities of antioxidant enzymes confirmed by histopathology. Lipid peroxidation level, IL-6, NO and CRP markedly increased compared to control. VCO supplementation prior to MTX injection attenuated MTX-induced oxidative nephrotoxicity via prominent increases in GSH and antioxidant enzyme activities in a dose-dependent manner. The renal inflammatory markers and MDA depleted considerably compared to MTX control group. Histopathological alterations were mitigated to confirm the biochemical indices. VCO supplementation demonstrates nephroprotective activity by attenuating MTX oxidative nephrotoxicity via antioxidant and anti-inflammatory activities in kidney. Our results suggested that VCO may benefit cancer patients on MTX chemotherapy against kidney injury. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Altered xanthine oxidase and N-acetyltransferase activity in obese children.

PubMed

Chiney, Manoj S; Schwarzenberg, Sarah J; Johnson, L'aurelle A

2011-07-01

It is well established that oxidative and conjugative enzyme activity differs between obese and healthy-weight adults. However, the effect of obesity on drug metabolism in children has not been studied extensively. This study examined whether obese and healthy-weight children vary with respect to oxidative enzyme activity of CYP1A2, xanthine oxidase (XO) and conjugative enzyme activity of N-acetyltransferase 2 (NAT2). In vivo CYP1A2, XO and NAT2 activity was assessed in obese (n= 9) and lean (n= 16) children between the ages of 6-10 years using caffeine (118.3 ml Coca Cola®) as probe. Urine samples were collected in 2-h increments over 8 h. Caffeine and metabolites were measured using LC/MS, and urinary metabolic ratios were determined based on reported methods. Sixteen healthy-weight and nine obese children were evaluated. XO activity was elevated in paediatric obese volunteers compared with non-obese paediatric volunteers (XO metabolic ratio of 0.7 ± 0.06 vs. 0.6 ± 0.06, respectively, 95% CI 0.046, 0.154, P < 0.001). NAT2 activity was fivefold higher in the obese (1 ± 0.4) as compared with non-obese children (0.2 ± 0.1), 95% CI 0.26, 1.34, P < 0.05. However, no difference was observed in CYP1A2 activity between the groups (95% CI -2.72, 0.12, P > 0.05). This study provides evidence that obese children have elevated XO and NAT2 enzyme activity when compared with healthy-weight controls. Further studies are needed to determine how this may impact the efficacy of therapeutic agents that may undergo metabolism by these enzymes. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.

Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction.

PubMed Central

Prudêncio, M; Eady, R R; Sawers, G

2001-01-01

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp(92) [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369-382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g( parallel)=2.12 (A( parallel)=0 mT) and g( perpendicular)=2.30 (A( perpendicular)=6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp(92)-->Asn (D92N) mutant showed no characteristic type 2 hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp(92)-->Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite. PMID:11139389

Prenatal ethanol exposure alters steroidogenic enzyme activity in newborn rat testes.

PubMed

Kelce, W R; Rudeen, P K; Ganjam, V K

1989-10-01

We have examined the in utero effects of ethanol exposure on testicular steroidogenesis in newborn male pups. Pregnant Sprague-Dawley rats were fed a liquid ethanol diet (35% ethanol-derived calories), a pair-fed isocaloric liquid diet, or a standard laboratory rat chow and water diet beginning on Day 12 of gestation and continuing through parturition. Although there were no significant differences in the enzymatic activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase or C17,20-lyase, the enzymatic activity of 17 alpha-hydroxylase was significantly (p less than 0.01) reduced (i.e., approximately 36%) in the ethanol-exposed pups compared to those from the pair-fed and chow treatment groups. This lesion in testicular steroidogenic enzyme activity in newborn male pups exposed to alcohol in utero was transient as 17 alpha-hydroxylase activity from the ethanol-exposed animals returned to control levels by postnatal Day 20 and remained at control levels through adulthood (postnatal Day 60). These data suggest that the suppression of the perinatal testosterone surge in male rats exposed to alcohol in utero and the associated long term demasculinizing effects of prenatal ethanol exposure might be the result of reduced testicular steroidogenic enzyme activity in the perinatal animal.

Long-term fertilization, but not warming, shifts rates of ectomycorrhizal nutrient cycling in Arctic tussock tundra.

NASA Astrophysics Data System (ADS)

Dunleavy, H.; Mack, M. C.

2017-12-01

The role of ectomycorrhizae (ECM) in Arctic nutrient cycling may be changing as temperature, nutrient availability, and ECM shrub abundance and size increase. A shift in ECM function has been proposed as a possible mechanism for shrub expansion. While several studies demonstrate a higher abundance of ECM as well as community compositional shifts in response to long-term experimental warming and fertilization, direct measurements of functional responses are missing. To understand the potential role of ECM in soil biogeochemical processes of the changing Arctic, we investigated the functional response of ECM to 30 years of summer warming and increased nutrient availability by measuring potential activities of extracellular enzymes associated with nitrogen (N) and phosphorous (P) acquisition on ECM root tips. We hypothesize ECM enzyme activities will be higher with warmer temperatures. Conversely, fertilization will lower ECM enzyme activities as N and P become less limiting to host plants. Preliminary results strongly support our latter hypothesis, but not the first. Warming decreased hydrolytic P-associated and labile N-associated enzyme activities on individual root tips (pmol/min/mm2 root tip) by 30% and 83%, respectively. However, warming increased ECM abundance and did not alter community-level activities (pmol/min/cm3 soil). Fertilization decreased hydrolytic and oxidative enzymatic activities on individual root tips by 34 to 80% as well as on a community level by 67 to 93%, even though ECM shrubs were almost monodominant. The combined effect of warming and fertilization decreased labile N-associated enzyme activity by 82%, but had little effect on oxidative and other hydrolytic enzyme activities. Although both warming and fertilization decreased root tip activities, reflecting a potential reduction in plant allocation to mycorrhizal nutrient acquisition, only fertilization lowered rates of ECM nutrient cycling. The indirect relationship between ECM abundance and individual root tip activity highlights the importance of measuring ECM function to assess the role of this symbiosis in nutrient cycling.

Isolated and mixed effects of diuron and its metabolites on biotransformation enzymes and oxidative stress response of Nile tilapia (Oreochromis niloticus).

PubMed

Felício, Andréia Arantes; Freitas, Juliane Silberschmidt; Scarin, Jéssica Bolpeti; de Souza Ondei, Luciana; Teresa, Fabrício Barreto; Schlenk, Daniel; de Almeida, Eduardo Alves

2018-03-01

Diuron is one of the most used herbicide in the world, and its field application has been particularly increased in Brazil due to the expansion of sugarcane crops. Diuron has often been detected in freshwater ecosystems and it can be biodegraded into three main metabolites in the environment, the 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU). Negative effects under aquatic biota are still not well established for diuron, especially when considering its presence in mixture with its different metabolites. In this study, we evaluated the effects of diuron alone or in combination with its metabolites, DCPMU, DCPU and 3,4-DCA on biochemical stress responses and biotransformation activity of the fish Oreochromis niloticus. Results showed that diuron and its metabolites caused significant but dispersed alterations in oxidative stress markers and biotransformation enzymes, except for ethoxyresorufin-O-deethylase (EROD) activity, that presented a dose-dependent increase after exposure to either diuron or its metabolites. Glutathione S-transferase (GST) activity was significant lower in gills after exposure to diuron metabolites, but not diuron. Diuron, DCPMU and DCA also decreased the multixenobiotic resistance (MXR) activity. Lipid peroxidation levels were increased in gill after exposure to all compounds, indicating that the original compound and diuron metabolites can induce oxidative stress in fish. The integration of all biochemical responses by the Integrated Biomarker Response (IBR) model indicated that all compounds caused significant alterations in O. niloticus, but DCPMU caused the higher alterations in both liver and gill. Our findings imply that diuron and its metabolites may impair the physiological response related to biotransformation and antioxidant activity in fish at field concentrations. Such alterations could interfere with the ability of aquatic animals to adapt to environments contaminated by agriculture. Copyright © 2017 Elsevier Inc. All rights reserved.

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205

Oxidative stress and spermatogenesis suppression in the testis of cadmium-treated Bombyx mori larvae.

PubMed

Yuan, Hongxia; Qin, Fenjv; Guo, Weiqiang; Gu, Huajie; Shao, Aihua

2016-03-01

Bombyx mori L. (B. mori) were exposed to cadmium chloride (CdCl2) incorporated in an artificial diet (0, 6.25, 12.5, 25, and 50 mg kg(-1)) throughout the larval stage. Changes in malondialdehyde (MDA) and reduced glutathione (GSH) contents and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as their corresponding messenger RNA (mRNA) levels in the testes of the fifth instar larvae were evaluated. Additionally, spermatozoon deformation in the testes was examined. Upon Cd treatment, the MDA content in the testes was significantly increased in a concentration-dependent manner. Cd-exposed larvae had increased levels of glutathione. Pearson's correlation analysis revealed that SOD and CAT activities were positively correlated (R (2) = 0.605, P = 0.017). The changing trends in the mRNA levels of these enzymes were not always consistent with those of enzymatic activities. Alterations in GSH-Px activities and mRNA levels were positively correlated (R (2) = 0.771, P < 0.01). Morphological analysis revealed that Cd deformed and affected the maturation of spermatozoa. Our results collectively support a relationship between Cd and alterations in the levels of antioxidant enzymes in B. mori testes.

Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

PubMed

Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

2016-03-17

L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

NASA Astrophysics Data System (ADS)

Wollenberg, Lance A.

Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover, applications of immobilized P450 enzyme constructs will also be used for monitoring protein-protein interaction and metabolite production with the use of immobilized-P450 bioreactor constructs. This work provides insight into the effect on catalytic activity caused by both P450 aggregation as well as isoform-specific protein-protein interactions and provides insight in the production of biosynthetically produced drug metabolites

Antioxidant treatment ameliorates experimental diabetes-induced depressive-like behaviour and reduces oxidative stress in brain and pancreas.

PubMed

Réus, Gislaine Z; Dos Santos, Maria Augusta B; Abelaira, Helena M; Titus, Stephanie E; Carlessi, Anelise S; Matias, Beatriz I; Bruchchen, Livia; Florentino, Drielly; Vieira, Andriele; Petronilho, Fabricia; Ceretta, Luciane B; Zugno, Alexandra I; Quevedo, João

2016-03-01

Studies have shown a relationship between diabetes mellitus (DM) and the development of major depressive disorder. Alterations in oxidative stress are associated with the pathophysiology of both diabetes mellitus and major depressive disorder. This study aimed to evaluate the effects of antioxidants N-acetylcysteine and deferoxamine on behaviour and oxidative stress parameters in diabetic rats. To this aim, after induction of diabetes by a single dose of alloxan, Wistar rats were treated with N-acetylcysteine or deferoxamine for 14 days, and then depressive-like behaviour was evaluated. Oxidative stress parameters were assessed in the prefrontal cortex, hippocampus, amygdala, nucleus accumbens and pancreas. Diabetic rats displayed depressive-like behaviour, and treatment with N-acetylcysteine reversed this alteration. Carbonyl protein levels were increased in the prefrontal cortex, hippocampus and pancreas of diabetic rats, and both N-acetylcysteine and deferoxamine reversed these alterations. Lipid damage was increased in the prefrontal cortex, hippocampus, amygdala and pancreas; however, treatment with N-acetylcysteine or deferoxamine reversed lipid damage only in the hippocampus and pancreas. Superoxide dismutase activity was decreased in the amygdala, nucleus accumbens and pancreas of diabetic rats. In diabetic rats, there was a decrease in catalase enzyme activity in the prefrontal cortex, amygdala, nucleus accumbens and pancreas, but an increase in the hippocampus. Treatment with antioxidants did not have an effect on the activity of antioxidant enzymes. In conclusion, animal model of diabetes produced depressive-like behaviour and oxidative stress in the brain and periphery. Treatment with antioxidants could be a viable alternative to treat behavioural and biochemical alterations induced by diabetes. Copyright © 2015 John Wiley & Sons, Ltd.

Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

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

Sultatos, L.G.; Kaushik, R.

2008-08-01

The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, thesemore » changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.« less

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.

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

PubMed

Bueno, Amy N; Shrestha, Rashmi K; Ronau, Judith A; Babar, Aditya; Sheedlo, Michael J; Fuchs, Julian E; Paul, Lake N; Das, Chittaranjan

2015-10-06

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

EPA Science Inventory

Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

Apoptosis: A Four-Week Laboratory Investigation for Advanced Molecular and Cellular Biology Students

ERIC Educational Resources Information Center

DiBartolomeis, Susan M.; Mone, James P.

2003-01-01

Over the past decade, apoptosis has emerged as an important field of study central to ongoing research in many diverse fields, from developmental biology to cancer research. Apoptosis proceeds by a highly coordinated series of events that includes enzyme activation, DNA fragmentation, and alterations in plasma membrane permeability. The detection…

Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, part I.

PubMed

Armstrong, Scott C; Cozza, Kelly L

2003-01-01

Pharmacokinetic drug-drug interactions with morphine, hydromorphone, and oxymorphone are reviewed in this column. Morphine is a naturally occurring opiate that is metabolized chiefly through glucuronidation by uridine diphosphate glucuronosyl transferase (UGT) enzymes in the liver. These enzymes produce an active analgesic metabolite and a potentially toxic metabolite. In vivo drug-drug interaction studies with morphine are few, but they do suggest that inhibition or induction of UGT enzymes could alter morphine and its metabolite levels. These interactions could change analgesic efficacy. Hydromorphone and oxymorphone, close synthetic derivatives of morphine, are also metabolized primarily by UGT enzymes. Hydromorphone may have a toxic metabolite similar to morphine. In vivo drug-drug interaction studies with hydromorphone and oxymorphone have not been done, so it is difficult to make conclusions with these drugs.

Cytochrome P450 monooxygenases: perspectives for synthetic application.

PubMed

Urlacher, Vlada B; Eiben, Sabine

2006-07-01

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

Glyphosate Adversely Affects Danio rerio Males: Acetylcholinesterase Modulation and Oxidative Stress.

PubMed

Lopes, Fernanda Moreira; Caldas, Sergiane Souza; Primel, Ednei Gilberto; da Rosa, Carlos Eduardo

2017-04-01

It has been demonstrated that glyphosate-based herbicides are toxic to animals. In the present study, reactive oxygen species (ROS) generation, antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO), as well as the activity and expression of the acetylcholinesterase (AChE) enzyme, were evaluated in Danio rerio males exposed to 5 or 10 mg/L of glyphosate for 24 and 96 h. An increase in ACAP in gills after 24 h was observed in the animals exposed to 5 mg/L of glyphosate. A decrease in LPO was observed in brain tissue of animals exposed to 10 mg/L after 24 h, while an increase was observed in muscle after 96 h. No significant alterations were observed in ROS generation. AChE activity was not altered in muscles or brains of animals exposed to either glyphosate concentration for 24 or 96 h. However, gene expression of this enzyme in the brain was reduced after 24 h and was enhanced in both brain and muscle tissues after 96 h. Thus, contrary to previous findings that had attributed the imbalance in the oxidative state of animals exposed to glyphosate-based herbicides to surfactants and other inert compounds, the present study demonstrated that glyphosate per se promotes this same effect in zebrafish males. Although glyphosate concentrations did not alter AChE activity, this study demonstrated for the first time that this molecule affects ache expression in male zebrafish D. rerio.

Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

PubMed

Vokřál, Ivan; Jirásko, Robert; Stuchlíková, Lucie; Bártíková, Hana; Szotáková, Barbora; Lamka, Jiří; Várady, Marián; Skálová, Lenka

2013-09-23

The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant). H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed. The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites. Copyright © 2013 Elsevier B.V. All rights reserved.

Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

PubMed

Mali, Aniket V; Bhise, Sunita S; Katyare, Surendra S; Hegde, Mahabaleshwar V

2018-01-01

Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

PubMed Central

Gebhardt, R; Mecke, D

1983-01-01

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells. Images Fig. 1. PMID:6138251

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

PubMed

Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

2016-11-15

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

Reduced activities of thiamine-dependent and cytochrome c oxidase enzymes in cerebral cortex of cattle affected by sulfur-induced polioencephalomalacia

PubMed Central

Amat, Samat; Hendrick, Steve; Moshynskyy, Igor; Simko, Elemir

2017-01-01

Sulfur-induced polioencephalomalacia (PEM) is an important disease affecting cattle in certain geographical regions. However, the pathogenesis of brain damage is not completely understood. We previously observed that excess dietary sulfur may influence thiamine status and altered thiamine metabolism may be involved in the pathogenesis of sulfur-induced PEM in cattle. In this study, we evaluated the activities of thiamine-dependent enzymes [α-ketogluterate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH)] and cytochrome c oxidase (COX) in the cerebral cortex of sulfur-induced PEM-affected cattle (n = 9) and clinically normal cattle (n = 8, each group) exposed to low or high dietary sulfur [LS = 0.30% versus HS = 0.67% sulfur on a dry matter (DM) basis]. Enzyme activities in PEM brains were measured from the brain tissue regions and examined using ultraviolent (UV) light illumination to show fluorescence or non-fluorescence regions. No gross changes under regular or UV light, or histopathological changes indicative of PEM were detected in the brains of cattle exposed to LS or HS diets. The PDH, α-KGDH, and COX activities did not differ between LS and HS brains, but all enzymes showed significantly lower (P < 0.05) activities in UV-positive region of PEM brains compared with LS and HS brains. The UV-negative regions of PEM brain had similar PDH activities to LS and HS brains, but the activities of α-KGDH and COX were significantly lower than in LS and HS brains. The results of this study suggest that reduced enzyme activities of brain PHD, α-KGDH, and COX are associated with the pathogenesis of sulfur-induced PEM. PMID:29081580

Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation

PubMed Central

Pao, Kuan-Chuan; Stanley, Mathew; Han, Cong; Lai, Yu-Chiang; Murphy, Paul; Balk, Kristin; Wood, Nicola T.; Corti, Olga; Corvol, Jean-Christophe; Muqit, Miratul M.K.; Virdee, Satpal

2016-01-01

E3 ligases represent an important class of enzymes, yet there are currently no chemical probes to profile their activity. We develop a new class of activity-based probe by reengineering of a ubiquitin-charged E2 conjugating enzyme and demonstrate their utility by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase Parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in Parkin activation. We also profile Parkin patient disease-associated mutations and strikingly demonstrate that they largely mediate their effect by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous Parkin activity revealing that endogenous Parkin is activated in neuronal cell lines (≥75 %) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-Parkin signalling as demonstrated by compatibility with Parkinson’s disease patient-derived samples. PMID:26928937

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Characterization of Active Site Residues of Nitroalkane Oxidase†

PubMed Central

Valley, Michael P.; Fenny, Nana S.; Ali, Shah R.; Fitzpatrick, Paul F.

2010-01-01

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

Characterization of active site residues of nitroalkane oxidase.

PubMed

Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

2010-06-01

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. 2009 Elsevier Inc. All rights reserved.

Calcium signaling in plant cells in microgravity

NASA Astrophysics Data System (ADS)

Kordyum, E.

Changes in the intracellular Ca 2 + concentration in altered gravity (microgravity and clinostating) evidence that Ca2 + signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in eighties, a review highlighting the performed research and the possible significance of such Ca 2 + changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumably specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2 + ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravis ensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tensionalterations in the physicochemical properties of the membranechanges in membrane permeability, ion transport, membrane-bound enzyme activity, etc.metabolism rearrangementsphysiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect, in the first place, cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca 2 + messenger system. Changes in Ca 2 + influx/efflux and possible pathways of Ca 2 + signaling in plant cell biochemical regulation in altered gravity are discussed.

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks׳ gestation in nulliparous women who subsequently develop preeclampsia.

PubMed

Mistry, Hiten D; Gill, Carolyn A; Kurlak, Lesia O; Seed, Paul T; Hesketh, John E; Méplan, Catherine; Schomburg, Lutz; Chappell, Lucy C; Morgan, Linda; Poston, Lucilla

2015-01-01

Preeclampsia is a pregnancy-specific condition affecting 2-7% of women and a leading cause of perinatal and maternal morbidity and mortality. Deficiencies of specific micronutrient antioxidant activities associated with copper, selenium, zinc, and manganese have previously been linked to preeclampsia at the time of disease. Our aims were to investigate whether maternal plasma micronutrient concentrations and related antioxidant enzyme activities are altered before preeclampsia onset and to examine the dependence on genetic variations in these antioxidant enzymes. Predisease plasma samples (15±1 weeks׳ gestation) were obtained from women enrolled in the international Screening for Pregnancy Endpoints (SCOPE) study who subsequently developed preeclampsia (n=244) and from age- and BMI-matched normotensive controls (n=472). Micronutrient concentrations were measured by inductively coupled plasma mass spectrometry; associated antioxidant enzyme activities, selenoprotein-P, ceruloplasmin concentration and activity, antioxidant capacity, and markers of oxidative stress were measured by colorimetric assays. Sixty-four tag-single-nucleotide polymorphisms (SNPs) within genes encoding the antioxidant enzymes and selenoprotein-P were genotyped using allele-specific competitive PCR. Plasma copper and ceruloplasmin concentrations were modestly but significantly elevated in women who subsequently developed preeclampsia (both P<0.001) compared to controls (median (IQR), copper, 1957.4 (1787, 2177.5) vs 1850.0 (1663.5, 2051.5) µg/L; ceruloplasmin, 2.5 (1.4, 3.2) vs 2.2 (1.2, 3.0) µg/ml). There were no differences in other micronutrients or enzymes between groups. No relationship was observed between genotype for SNPs and antioxidant enzyme activity. This analysis of a prospective cohort study reports maternal micronutrient concentrations in combination with associated antioxidant enzymes and SNPs in their encoding genes in women at 15 weeks׳ gestation that subsequently developed preeclampsia. The modest elevation in copper may contribute to oxidative stress, later in pregnancy, in those women that go on to develop preeclampsia. The lack of evidence to support the hypothesis that functional SNPs influence antioxidant enzyme activity in pregnant women argues against a role for these genes in the etiology of preeclampsia. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Mutations affecting gyrase in Haemophilus influenzae.

PubMed Central

Setlow, J K; Cabrera-Juárez, E; Albritton, W L; Spikes, D; Mutschler, A

1985-01-01

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch. PMID:2997115

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants

PubMed Central

Renault, Hugues; De Marothy, Minttu; Jonasson, Gabriella; Lara, Patricia; Nelson, David R.; Nilsson, IngMarie; André, François; von Heijne, Gunnar; Werck-Reichhart, Danièle

2017-01-01

Abstract Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity. PMID:28505373

Use of ferrous iron by metallo-β-lactamases.

PubMed

Cahill, Samuel T; Tarhonskaya, Hanna; Rydzik, Anna M; Flashman, Emily; McDonough, Michael A; Schofield, Christopher J; Brem, Jürgen

2016-10-01

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

PubMed

Renault, Hugues; De Marothy, Minttu; Jonasson, Gabriella; Lara, Patricia; Nelson, David R; Nilsson, IngMarie; André, François; von Heijne, Gunnar; Werck-Reichhart, Danièle

2017-08-01

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator

PubMed Central

Elcombe, Clifford R.; Peffer, Richard C.; Wolf, Douglas C.; Bailey, Jason; Bars, Remi; Bell, David; Cattley, Russell C.; Ferguson, Stephen S.; Geter, David; Goetz, Amber; Goodman, Jay I.; Hester, Susan; Jacobs, Abigail; Omiecinski, Curtis J.; Schoeny, Rita; Xie, Wen; Lake, Brian G.

2014-01-01

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk. PMID:24180433

Trans unsaturated fatty acids inhibit lecithin: cholesterol acyltransferase and alter its positional specificity.

PubMed

Subbaiah, P V; Subramanian, V S; Liu, M

1998-07-01

Although dietary trans unsaturated fatty acids (TUFA) are known to decrease plasma HDL, the underlying mechanisms for this effect are unclear. We tested the hypothesis that the decreased HDL is due to an inhibition of lecithin:cholesterol acyltransferase (LCAT), the enzyme essential for the formation of HDL, by determining the activity of purified LCAT in the presence of synthetic phosphatidylcholine (PC) substrates containing TUFA. Both human and rat LCATs exhibited significantly lower activity (-37% to -50%) with PCs containing 18:1t or 18:2t, when compared with the PCs containing corresponding cis isomers. TUFA-containing PCs also inhibited the enzyme activity competitively, when added to egg PC substrate. The inhibition of LCAT activity was not due to changes in the fluidity of the substrate particle. However, the inhibition depended on the position occupied by TUFA in the PC, as well as on the paired fatty acid. Thus, for human LCAT, 18:1t was more inhibitory when present at sn-2 position of PC, than at sn-1, when paired with 16:0. In contrast, when paired with 20:4, 18:1t was more inhibitory at sn-1 position of PC. Both human and rat LCATs, which are normally specific for the sn-2 acyl group of PC, exhibited an alteration in their positional specificity when 16:0-18:1t PC or 16:1t-20:4 PC was used as substrate, deriving 26-86% of the total acyl groups for cholesterol esterification from the sn-1 position. These results show that the trans fatty acids decrease high density lipoprotein through their inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, and also alter LCAT's positional specificity, inducing the formation of more saturated cholesteryl esters, which are more atherogenic.

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

PubMed

Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; van der Horst, Sjors; Theelen, Bart; de Vries, Ronald P; van den Brink, Joost

2016-01-01

Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.

Vascular effects of advanced glycation endproducts: Clinical effects and molecular mechanisms☆

PubMed Central

Stirban, Alin; Gawlowski, Thomas; Roden, Michael

2013-01-01

The enhanced generation and accumulation of advanced glycation endproducts (AGEs) have been linked to increased risk for macrovascular and microvascular complications associated with diabetes mellitus. AGEs result from the nonenzymatic reaction of reducing sugars with proteins, lipids, and nucleic acids, potentially altering their function by disrupting molecular conformation, promoting cross-linking, altering enzyme activity, reducing their clearance, and impairing receptor recognition. AGEs may also activate specific receptors, like the receptor for AGEs (RAGE), which is present on the surface of all cells relevant to atherosclerotic processes, triggering oxidative stress, inflammation and apoptosis. Understanding the pathogenic mechanisms of AGEs is paramount to develop strategies against diabetic and cardiovascular complications. PMID:24634815

AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

PubMed

Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

2006-05-01

The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability.

Relationship between Advanced Glycation End Products and Steroidogenesis in PCOS.

PubMed

Garg, Deepika; Merhi, Zaher

2016-10-21

Women with PCOS have elevated levels of the harmful Advanced Glycation End Products (AGEs), which are highly reactive molecules formed after glycation of lipids and proteins. Additionally, AGEs accumulate in the ovaries of women with PCOS potentially contributing to the well-documented abnormal steroidogenesis and folliculogenesis. A systematic review of articles and abstracts available in PubMed was conducted and presented in a systemic manner. This article reports changes in steroidogenic enzyme activity in granulosa and theca cells in PCOS and PCOS-models. It also described the changes in AGEs and their receptors in the ovaries of women with PCOS and presents the underlying mechanism(s) whereby AGEs could be responsible for the PCOS-related changes in granulosa and theca cell function thus adversely impacting steroidogenesis and follicular development. AGEs are associated with hyperandrogenism in PCOS possibly by altering the activity of various enzymes such as cholesterol side-chain cleavage enzyme cytochrome P450, steroidogenic acute regulatory protein, 17α-hydroxylase, and 3β-hydroxysteroid dehydrogenase. AGEs also affect luteinizing hormone receptor and anti-Mullerian hormone receptor expression as well as their signaling pathways in granulosa cells. A better understanding of how AGEs alter granulosa and theca cell function is likely to contribute meaningfully to a conceptual framework whereby new interventions to prevent and/or treat ovarian dysfunction in PCOS can ultimately be developed.

Hepatic and renal oxidative stress in acute toxicity of N-nitrosodiethylamine in rats.

PubMed

Bansal, A K; Trivedi, R; Soni, G L; Bhatnagar, D

2000-09-01

Nitrosoamines such as N-nitrosodiethylamine (NDEA) produce oxidative stress due to generation of reactive oxygen species and may alter antioxidant defence system in the tissues. NDEA was administered ip as a single dose to rats in LD50 or in lower amounts and the animals were sacrificed after 0-48 hr of treatment. The results showed that lipid peroxidation in liver increased, however no significant increase in kidney LPO was observed after NDEA administration. Superoxide dismutase (SOD) and glutathione reductase (GSH-R) activity increased in liver, however, catalase (CAT) activity in liver was inhibited in NDEA treated rats. Kidney showed an increase in SOD activity after an initial decrease along with increase in GSH-R activity in NDEA treated rats. However, kidney CAT activity was not significantly altered in NDEA intoxicated rats. Serum transaminases, serum alkaline phosphatase blood urea nitrogen, serum creatinine and scrum proteins were elevated in NDEA treated rats. The results indicate NDEA-induced oxidative stress and alteration in antioxidant enzymes in liver and kidney to neutralise oxidative stress.

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii*

PubMed Central

Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H.; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D.

2014-01-01

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

Ethanol induced hepatic mitochondrial dysfunction is attenuated by all trans retinoic acid supplementation.

PubMed

Nair, Saritha S; Prathibha, P; Rejitha, S; Indira, M

2015-08-15

Alcoholics have reduced vitamin A levels in serum since vitamin A and ethanol share the same metabolic pathway. Vitamin A supplementation has an additive effect on ethanol induced toxicity. Hence in this study, we assessed the impact of supplementation of all trans retinoic acid (ATRA), an active metabolite of vitamin A on ethanol induced disruptive alterations in liver mitochondria. Male Sprague Dawley rats were grouped as follows: I: Control; II: Ethanol (4 g/kg b.wt./day); III: ATRA (100 μg/kg b.wt./day); and IV: Ethanol (4 g/kg b.wt./day)+ATRA (100 μg/kg b.wt./day). Duration of the experiment was 90 days, after which the animals were sacrificed for the study. The key enzymes of energy metabolism, reactive oxygen species, mitochondrial membrane potential and hepatic mRNA expressions of Bax, Bcl-2, c-fos and c-jun were assessed. Ethanol administration increased the reactive oxygen species generation in mitochondria. It also decreased the activities of the enzymes of citric acid cycle and oxidative phosphorylation. ATP content and mitochondrial membrane potential were decreased and cytosolic cytochrome c was increased consequently enhancing apoptosis. All these alterations were altered significantly on ATRA supplementation along with ethanol. These results were reinforced by our histopathological studies. ATRA supplementation to ethanol fed rats, led to reduction in oxidative stress, decreased calcium overload in the matrix and increased mitochondrial membrane potential, which might have altered the mitochondrial energy metabolism and elevated ATP production thereby reducing the apoptotic alterations. Hence ATRA supplementation seemed to be an effective intervention against alcohol induced mitochondrial dysfunction. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

PubMed Central

Bolton, P. G.; Dean, A. C. R.

1972-01-01

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined. PMID:4342213

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney.

PubMed

Priyamvada, Shubha; Khan, Sara A; Khan, Md Wasim; Khan, Sheeba; Farooq, Neelam; Khan, Farah; Yusufi, A N K

2010-01-01

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism. Copyright 2009 Elsevier Ltd. All rights reserved.

[The effect of cytostatic therapy with vincristin sulphate on disaccarchidases of rat intestinal mucosa (author's transl)].

PubMed

Hartwich, G; Leicher, H; Müller, H; Domschke, W; Matzkies, F

1976-01-01

This report shows that appropriate doses of vincristin sulphate may decrease disaccharidase activities of intestinal mucosa. With the higher doses of the cytostatic drug, the drastic drop of enzyme activities is associated with morphological alterations of the mucosa; disacchardiase activities remain depressed at least for a couple of days even after full morphological restoration of the mucosa. Studies in man should reveal whether similar intestinal lesions occur due to therapeutic doses of vincristin sulphate.

Changes in substrate availability drive carbon cycle response to chronic warming

DOE PAGES

Pold, Grace; Grandy, A. Stuart; Melillo, Jerry M.; ...

2017-03-22

As earth's climate continues to warm, it is important to understand how the capacity of terrestrial ecosystems to retain carbon (C) will be affected. We combined measurements of microbial activity with the concentration, quality, and physical accessibility of soil carbon to microorganisms to evaluate the mechanisms by which more than two decades of experimental warming has altered the carbon cycle in a Northeast US temperate deciduous forest. We have found that concentrations of soil organic matter were reduced in both the organic and mineral soil horizons. The molecular composition of the carbon was altered in the mineral soil with significantmore » reductions in the relative abundance of polysaccharides and lignin, and an increase in lipids. Mineral-associated organic matter was preferentially depleted by warming in the top 3 cm of mineral soil. We found that potential extracellular enzyme activity per gram of soil at a common temperature was generally unaffected by warming treatment. However, by measuring potential extracellular enzyme activities between 4 and 30 °C, we found that activity per unit microbial biomass at in-situ temperatures was increased by warming. This was associated with a tendency for microbial biomass to decrease with warming. These results indicate that chronic warming has reduced soil organic matter concentrations, selecting for a smaller but more active microbial community increasingly dependent on mineral-associated organic matter.« less

Deletion of murine choline dehydrogenase results in diminished sperm motility

PubMed Central

Johnson, Amy R.; Craciunescu, Corneliu N.; Guo, Zhong; Teng, Ya-Wen; Thresher, Randy J.; Blusztajn, Jan K.; Zeisel, Steven H.

2010-01-01

Choline dehydrogenase (CHDH) catalyzes the conversion of choline to betaine, an important methyl donor and organic osmolyte. We have previously identified single nucleotide polymorphisms (SNPs) in the human CHDH gene that, when present, seem to alter the activity of the CHDH enzyme. These SNPs occur frequently in humans. We created a Chdh−/− mouse to determine the functional effects of mutations that result in decreased CHDH activity. Chdh deletion did not affect fetal viability or alter growth or survival of these mice. Only one of eleven Chdh−/− males was able to reproduce. Loss of CHDH activity resulted in decreased testicular betaine and increased choline and PCho concentrations. Chdh+/+ and Chdh−/− mice produced comparable amounts of sperm; the impaired fertility was due to diminished sperm motility in the Chdh−/− males. Transmission electron microscopy revealed abnormal mitochondrial morphology in Chdh−/− sperm. ATP content, total mitochondrial dehydrogenase activity and inner mitochondrial membrane polarization were all significantly reduced in sperm from Chdh−/− animals. Mitochondrial changes were also detected in liver, kidney, heart, and testis tissues. We suggest that men who have SNPs in CHDH that decrease the activity of the CHDH enzyme could have decreased sperm motility and fertility.—Johnson, A. R., Craciunescu, C. N., Guo, Z., Teng, Y.-W., Thresher, R. J., Blusztajn, J. K., Zeisel, S. H. Deletion of murine choline dehydrogenase results in diminished sperm motility. PMID:20371614

Dietary supplementation of tiger nut alters biochemical parameters relevant to erectile function in l-NAME treated rats.

PubMed

Olabiyi, Ayodeji A; Carvalho, Fabiano B; Bottari, Nathieli B; Lopes, Thauan F; da Costa, Pauline; Stefanelo, Naiara; Morsch, Vera M; Akindahunsi, Afolabi A; Oboh, Ganiyu; Schetinger, Maria Rosa

2018-07-01

Tiger nut tubers have been reportedly used for the treatment of erectile dysfunction (ED) in folk medicine without scientific basis. Hence, this study evaluated the effect of tiger nut on erectile dysfunction by assessing biochemical parameters relevant to ED in male rats by nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) treatment. Rats were divided into five groups (n = 10) each: Control group; l-NAME plus basal diet; l-NAME plus Sildenafil citrate; diet supplemented processed tiger nut (20%) plus l-NAME;diet supplemented raw tiger nut (20%) plus l-NAME. l-NAME pre-treatment (40 mg/kg/day) lasted for 14 days. Arginase, acetycholinesterase (AChE) and adenosine deaminase (ADA) activities as well as nitric oxide levels (NO) in serum, brain and penile tissue were measured. l-NAME increased the activity of arginase, AChE and ADA and reduced NO levels. However, dietary supplementation with tiger nut caused a reduction on the activities of the above enzymes and up regulated nitric oxide levels when compared to the control group. The effect of tiger nut supplemented diet may be said to prevent alterations of the activities of the enzymes relevant in erectile function. Quercetin was revealed to be the most active component of tiger nut tuber by HPLC finger printing. Copyright © 2018. Published by Elsevier Ltd.

Changes in substrate availability drive carbon cycle response to chronic warming

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

Pold, Grace; Grandy, A. Stuart; Melillo, Jerry M.

As earth's climate continues to warm, it is important to understand how the capacity of terrestrial ecosystems to retain carbon (C) will be affected. We combined measurements of microbial activity with the concentration, quality, and physical accessibility of soil carbon to microorganisms to evaluate the mechanisms by which more than two decades of experimental warming has altered the carbon cycle in a Northeast US temperate deciduous forest. We have found that concentrations of soil organic matter were reduced in both the organic and mineral soil horizons. The molecular composition of the carbon was altered in the mineral soil with significantmore » reductions in the relative abundance of polysaccharides and lignin, and an increase in lipids. Mineral-associated organic matter was preferentially depleted by warming in the top 3 cm of mineral soil. We found that potential extracellular enzyme activity per gram of soil at a common temperature was generally unaffected by warming treatment. However, by measuring potential extracellular enzyme activities between 4 and 30 °C, we found that activity per unit microbial biomass at in-situ temperatures was increased by warming. This was associated with a tendency for microbial biomass to decrease with warming. These results indicate that chronic warming has reduced soil organic matter concentrations, selecting for a smaller but more active microbial community increasingly dependent on mineral-associated organic matter.« less

Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

PubMed

Lončar, Nikola; Fraaije, Marco W

2015-03-01

Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

Antioxidant Properties and Cardioprotective Mechanism of Malaysian Propolis in Rats.

PubMed

Ahmed, Romana; Tanvir, E M; Hossen, Md Sakib; Afroz, Rizwana; Ahmmed, Istiyak; Rumpa, Nur-E-Noushin; Paul, Sudip; Gan, Siew Hua; Sulaiman, Siti Amrah; Khalil, Md Ibrahim

2017-01-01

Propolis contains high concentrations of polyphenols, flavonoids, tannins, ascorbic acid, and reducing sugars and proteins. Malaysian Propolis (MP) has been reported to exhibit high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and ferric reducing antioxidant power (FRAP) values. Herein, we report the antioxidant properties and cardioprotective properties of MP in isoproterenol- (ISO-) induced myocardial infarction in rats. Male Wistar rats ( n = 32) were pretreated orally with an ethanol extract of MP (100 mg/kg/day) for 30 consecutive days. Subcutaneous injection of ISO (85 mg/kg in saline) for two consecutive days caused a significant increase in serum cardiac marker enzymes and cardiac troponin I levels and altered serum lipid profiles. In addition significantly increased lipid peroxides and decreased activities of cellular antioxidant defense enzymes were observed in the myocardium. However, pretreatment of ischemic rats with MP ameliorated the biochemical parameters, indicating the protective effect of MP against ISO-induced ischemia in rats. Histopathological findings obtained for the myocardium further confirmed the biochemical findings. It is concluded that MP exhibits cardioprotective activity against ISO-induced oxidative stress through its direct cytotoxic radical-scavenging activities. It is also plausible that MP contributed to endogenous antioxidant enzyme activity via inhibition of lipid peroxidation.

Phytochemicals as Anticancer and Chemopreventive Topoisomerase II Poisons

PubMed Central

Ketron, Adam C.

2013-01-01

Phytochemicals are a rich source of anticancer drugs and chemopreventive agents. Several of these chemicals appear to exert at least some of their effects through interactions with topoisomerase II, an essential enzyme that regulates DNA supercoiling and removes knots and tangles from the genome. Topoisomerase II-active phytochemicals function by stabilizing covalent protein-cleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme. As a result, these compounds convert topoisomerase II to a cellular toxin that fragments the genome. Because of their mode of action, they are referred to as topoisomerase II poisons as opposed to catalytic inhibitors. The first sections of this article discuss DNA topology, the catalytic cycle of topoisomerase II, and the two mechanisms (interfacial vs. covalent) by which different classes of topoisomerase II poisons alter enzyme activity. Subsequent sections discuss the effects of several phytochemicals on the type II enzyme, including demethyl-epipodophyllotoxins (semisynthetic anticancer drugs) as well as flavones, flavonols, isoflavones, catechins, isothiocyanates, and curcumin (dietary chemopreventive agents). Finally, the leukemogenic potential of topoisomerase II-targeted phytochemicals is described. PMID:24678287

Impaired in vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy.

PubMed

Dodd, Michael S; Atherton, Helen J; Carr, Carolyn A; Stuckey, Daniel J; West, James A; Griffin, Julian L; Radda, George K; Clarke, Kieran; Heather, Lisa C; Tyler, Damian J

2014-11-01

Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment. © 2014 American Heart Association, Inc.

Impaired In Vivo Mitochondrial Krebs Cycle Activity After Myocardial Infarction Assessed Using Hyperpolarized Magnetic Resonance Spectroscopy

PubMed Central

Carr, Carolyn A.; Stuckey, Daniel J.; West, James A.; Griffin, Julian L.; Radda, George K.; Clarke, Kieran; Heather, Lisa C.; Tyler, Damian J.

2015-01-01

Background Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. Methods and Results Using hyperpolarized carbon-13 (13C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased 13C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. Conclusions The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment. PMID:25201905

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

PubMed

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

2017-04-01

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

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.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice

PubMed Central

Burns, David P.; Ali, Izza; Rieux, Clement; Healy, James; Jasionek, Greg; O’Halloran, Ken D.

2017-01-01

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease. PMID:29210997

Salivary defense system alters in vegetarian

PubMed Central

Amirmozafari, Nour; Pourghafar, Houra; Sariri, Reyhaneh

2013-01-01

Purpose The aim of this research was investigating antimicrobial and enzymatic antioxidant activities in salivary fluids of vegetarians as compared to normal subjects. Material & Methods Antimicrobial activity of the saliva samples was evaluated against four clinically important bacteria. The biological activities of three of the main antioxidant enzymes of saliva were measured using appropriate methods of enzyme assay in both groups. Results According to the results, saliva obtained from vegetarians showed a reduced inhibitory effect on growth of Staphylococcus aureus, Klebsiella oxytoca, Pseudomonas aeruginosa and Escherichia coli as compared to those obtained from the non-vegetarian subjects. The activity of salivary peroxidase, catalase and superoxide dismutase showed a statistically marked decrease in vegetarian group. Conclusions According to our literature survey, this is the first report on the antibacterial and antioxidant capacity in saliva of vegetarians. Results obtained from the present study have opened a new line of research with the basis of saliva as a research tool. PMID:25737889

The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers

PubMed Central

Russler-Germain, David A.; Spencer, David H.; Young, Margaret A.; Lamprecht, Tamara L.; Miller, Christopher A.; Fulton, Robert; Meyer, Matthew R.; Erdmann-Gilmore, Petra; Townsend, R. Reid; Wilson, Richard K.; Ley, Timothy J.

2014-01-01

Summary Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ~30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ~80% compared to the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity but co-expression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes. PMID:24656771

Inhibition effect of food preservatives on endoproteinases.

PubMed

Esimbekova, Elena N; Asanova, Anastasiya A; Deeva, Anna A; Kratasyuk, Valentina A

2017-11-15

The present manuscript proposes a novel approach to assess the impact of food additives on human metabolism by analysing their effect on biomarker enzyme activity. Alterations in the activity of pancreatic enzymes, such as chymotrypsin and trypsin, which are affected by the most common food preservatives, sodium benzoate (E211), potassium sorbate (E202) and sorbic acid (E200), have been evaluated. The proteinase activity was analysed with a bioluminescent method using the light intensity decay constant. Our study revealed that the preservatives reduce proteinase activity by 50% (EC 50 ) at a much lower concentration than their acceptable daily intake (ADI). Thus, sodium benzoate and sorbic acid have an inhibition effect on chymotrypsin at concentrations 14 times lower and 70 times lower than their ADI and this increases with exposure time. Food preservative consumption impacts negatively on protein digestion, which is especially dangerous for patients with pancreatitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

PubMed Central

Hossain, Mohammed Iqbal; Roulston, Carli Lorraine; Stapleton, David Ian

2014-01-01

Background Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. Results Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. Conclusion Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity. PMID:24858129

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering.

PubMed

Nagao, Jun-ichi; Aso, Yuji; Shioya, Kouki; Nakayama, Jiro; Sonomoto, Kenji

2007-01-01

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

PubMed

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

PubMed

Terentis, Andrew C; Freewan, Mohammed; Sempértegui Plaza, Tito S; Raftery, Mark J; Stocker, Roland; Thomas, Shane R

2010-01-26

The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory role by catalyzing the oxidative degradation of l-tryptophan. Here we show that the selenezal drug ebselen is a potent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a manner independent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDO (rIDO) with an apparent inhibition constant of 94 +/- 17 nM. Optical and resonance Raman spectroscopy showed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron from the predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch of the CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed that ebselen enhanced nonproductive l-tryptophan binding, while circular dichroism indicated that the drug reduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed that ebselen reacted with multiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitol reversed the effects of the drug on the heme environment and significantly restored enzyme activity. These findings indicate that ebselen inhibits IDO activity by reacting with the enzyme's cysteine residues that result in changes to protein conformation and active site heme, leading to an increase in the level of nonproductive substrate binding. This study highlights that modification of cysteine residues is a novel and effective means of inhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents a previously unrecognized in vivo target of ebselen.

Counteraction of the Antiapoptotic Protein Survivin by Diverting Expression to its Proapoptotic Splice Variant Survivin-2B

DTIC Science & Technology

2010-01-01

negatively regulated by low glucose. [17] Further, glucose restriction activates the longevity- associated histone and protein deacetylase, SIRT1 ...two glycolytic enzymes it activates (aldolase A and pyruvate kinase M2 ) by altering Sp1’s phosphorylation state; that is, glucose promotes...pattern of decreasing with low glucose. We also studied SIRT1 because it was already reported to epigenetically silence survivin transcription and

Metabolomic, enzymatic, and histochemical analyzes of cassava roots during postharvest physiological deterioration.

PubMed

Uarrota, Virgílio Gavicho; Maraschin, Marcelo

2015-11-05

Under postharvest physiological deterioration cassava root tubers alter the expression of biosynthetic pathways of certain primary and secondary metabolites, as well as the activity of some scavenging enzymes. Therefore, in this study we hypothesized that cassava cultivars differ as to their physiological responses to deterioration and their biochemical profiles can be an indicative of the tolerance or susceptibility to deterioration. The results corroborate the working hypothesis, revealing that high Levels of phenolic acids, scopoletin, carotenoids, proteins, and augmented activities of guaiacol peroxidase and hydrogen peroxide in non-stored cassava roots can be used as potential biomarkers of cassava deterioration. Cassava physiological deterioration depends on cultivar and many compounds are up and downregulated during storage time. Secondary metabolites, enzymes, scopoletin, scavenging reactive oxygen species, and acidic polysaccharides are activated as responses to the physiological stress induced in root tubers.

Evaluation of metabolic enzymes in response to Excel Mera 71, a glyphosate-based herbicide, and recovery pattern in freshwater teleostean fishes.

PubMed

Samanta, Palas; Pal, Sandipan; Mukherjee, Aloke Kumar; Ghosh, Apurba Ratan

2014-01-01

Metabolic enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated in Indian teleostean fishes, namely, Anabas testudineus (Bloch) and Heteropneustes fossilis (Bloch), for an exposure to 30 days of Excel Mera 71 (17.2 mg/L), a glyphosate formulation, and subsequent depuration under Liv.52, a plant extract at a dose of 187.5 mg/d/250 L for the same period in the same tissues under laboratory condition. ALT activity was significantly increased (P<0.05) in all the tissues and raised up to 229.19% in liver of A. testudineus (229.19%) and 128.61% in liver of H. fossilis. AST also increased significantly (P<0.05) and was maximum in liver of H. fossilis (526.19%) and minimum in gill of A. testudineus (124.38%). ALP activity was also raised highly in intestine of H. fossilis (490.61%) but was less in kidney of H. fossilis (149.48%). The results indicated that Excel Mera 71 caused alterations in the metabolic enzymatic activities in fish tissues and AST showed the highest alteration in both the fishes, while lowest in ALP and ALT in A. testudineus and H. fossilis, respectively. During depuration under Liv.52, all the enzyme activities came down towards the control condition which indicated the compensatory response by the fish against this herbicidal stress and it was in the following order: AST>ALT>ALP, in A. testudineus, while H. fossilis showed the following trend: ALT>AST>ALP. Therefore, these parameters could be used as indicators of herbicidal pollution in aquatic organisms and were recommended for environmental monitoring for investigating the mechanism involved in the recovery pattern.

Glutathione metabolic status in the aged rabbit aorta.

PubMed

Lapenna, Domenico; Ciofani, Giuliano; Giamberardino, Maria Adele

2017-05-01

It is not known whether aging alters glutathione metabolic status of the mammalian arterial tissue favoring vascular oxidative stress and dysfunction. Thus we assessed total, reduced and oxidized glutathione (TG, GSH and GSSG, respectively), the glutathione redox ratio (GRR, namely [GSSG]/[GSH+2GSSG]×100), and the activities of the glutathione status-regulating enzymes glutathione reductase (GSSG-Red), γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyl transpeptidase (γ-GT) in the aortic tissue of 9 young adult control rabbits (YACR, about 4months old) and 9 aged rabbits (AR, about 4.5years old); aortic lipid and protein oxidation and H 2 O 2 were also determined as oxidative stress indicators. Vascular function was assessed on aortic ring preparations. TG and GSH concentrations, together with γ-GCS and γ-GT activities, were significantly lower, while GSSG content and the GRR higher, in the AR than in the YACR aortas; GSSG-Red activity did not differ significantly between the two groups. Heightened levels of lipid and protein oxidation and H 2 O 2 occurred in the AR aortas, indicating age-dependent vascular oxidative stress. Moreover, in the whole population of 18 rabbits, the aortic values of GSH and related enzyme activities were inversely and significantly correlated with those of lipid and protein oxidation and H 2 O 2 , highlighting the antioxidant role of GSH and related enzymes in the vascular tissue. Aortic endothelium-dependent vasodilation was lower in the AR than in the YACR. In conclusion, glutathione metabolic status is altered in the aged rabbit aorta reflecting depressed γ-GCS- and γ-GT-related GSH biosynthesis and GSSG burden eventually favoring vascular oxidative stress and dysfunction. Copyright © 2017 Elsevier Inc. All rights reserved.

Function and Dynamics of Auxin and Carbohydrates during Earlywood/Latewood Transition in Scots Pine1

PubMed Central

Uggla, Claes; Magel, Elisabeth; Moritz, Thomas; Sundberg, Björn

2001-01-01

In temperate regions the annual pattern of wood development is characterized by the formation of radially narrow and thick walled latewood cells. This takes place at the later part of the growing season when cambial cell division declines. To gain new insight into the regulation of this process, micro-analytical techniques were used to visualize the distribution of indole-3-acetic acid (IAA), soluble carbohydrates, and activities of sucrose (Suc)-metabolizing enzymes across the cambial region tissues in Scots pine (Pinus sylvestris). The total amount of IAA in the cambial region did not change with latewood initiation. But its radial distribution pattern was altered, resulting in an increased concentration in the cambial meristem and its recent derivatives. Thus, initiation of latewood formation and cessation of cambial cell division is not a consequence of decreased IAA concentrations in dividing and expanding cells. Rather, IAA most likely has a role in defining the altered developmental pattern associated with latewood formation. Carbohydrates and enzyme activities showed distinctive radial distribution patterns. Suc peaked in the phloem and decreased sharply to low levels across the cambial zone, whereas fructose and glucose reached their highest levels in the maturing tracheids. Suc synthase was the dominating Suc cleaving enzyme with a peak in the secondary wall-forming tracheids and in the phloem. Soluble acid invertase peaked in dividing and expanding cells. Suc-phosphate synthase had its highest activities in the phloem. Activities of cell wall bound invertase were low. The absence of major seasonal variations indicates that carbohydrate availability is not a trigger for latewood initiation. However, steep concentration gradients of the sugars suggest a role for sugar signaling in vascular development. PMID:11299382

Function and dynamics of auxin and carbohydrates during earlywood/latewood transition in scots pine.

PubMed

Uggla, C; Magel, E; Moritz, T; Sundberg, B

2001-04-01

In temperate regions the annual pattern of wood development is characterized by the formation of radially narrow and thick walled latewood cells. This takes place at the later part of the growing season when cambial cell division declines. To gain new insight into the regulation of this process, micro-analytical techniques were used to visualize the distribution of indole-3-acetic acid (IAA), soluble carbohydrates, and activities of sucrose (Suc)-metabolizing enzymes across the cambial region tissues in Scots pine (Pinus sylvestris). The total amount of IAA in the cambial region did not change with latewood initiation. But its radial distribution pattern was altered, resulting in an increased concentration in the cambial meristem and its recent derivatives. Thus, initiation of latewood formation and cessation of cambial cell division is not a consequence of decreased IAA concentrations in dividing and expanding cells. Rather, IAA most likely has a role in defining the altered developmental pattern associated with latewood formation. Carbohydrates and enzyme activities showed distinctive radial distribution patterns. Suc peaked in the phloem and decreased sharply to low levels across the cambial zone, whereas fructose and glucose reached their highest levels in the maturing tracheids. Suc synthase was the dominating Suc cleaving enzyme with a peak in the secondary wall-forming tracheids and in the phloem. Soluble acid invertase peaked in dividing and expanding cells. Suc-phosphate synthase had its highest activities in the phloem. Activities of cell wall bound invertase were low. The absence of major seasonal variations indicates that carbohydrate availability is not a trigger for latewood initiation. However, steep concentration gradients of the sugars suggest a role for sugar signaling in vascular development.

Contrasting effects of biochar versus manure on soil microbial communities and enzyme activities in an Aridisol.

PubMed

Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A; Lentz, Rodrick D

2016-01-01

Biochar can increase microbial activity, alter microbial community structure, and increase soil fertility in arid and semi-arid soils, but at relatively high rates that may be impractical for large-scale field studies. This contrasts with organic amendments such as manure, which can be abundant and inexpensive if locally available, and thus can be applied to fields at greater rates than biochar. In a field study comparing biochar and manure, a fast pyrolysis hardwood biochar (22.4 Mg ha(-1)), dairy manure (42 Mg ha(-1) dry wt), a combination of biochar and manure at the aforementioned rates, or no amendment (control) was applied to an Aridisol (n=3) in fall 2008. Plots were annually cropped to corn (Zea maize L.). Surface soils (0-30 cm) were sampled directly under corn plants in late June 2009 and early August 2012, and assayed for microbial community fatty acid methyl ester (FAME) profiles and six extracellular enzyme activities involved in soil C, N, and P cycling. Arbuscular mycorrhizal (AM) fungal colonization was assayed in corn roots in 2012. Biochar had no effect on microbial biomass, community structure, extracellular enzyme activities, or AM fungi root colonization of corn. In the short-term, manure amendment increased microbial biomass, altered microbial community structure, and significantly reduced the relative concentration of the AM fungal biomass in soil. Manure also reduced the percent root colonization of corn by AM fungi in the longer-term. Thus, biochar and manure had contrasting short-term effects on soil microbial communities, perhaps because of the relatively low application rate of biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase.

PubMed

Fung, Shin Yee; Lee, Mui Li; Tan, Nget Hong

2015-03-01

Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

Drug Metabolism and Transport During Pregnancy: How Does Drug Disposition Change during Pregnancy and What Are the Mechanisms that Cause Such Changes?

PubMed Central

Thummel, Kenneth E.

2013-01-01

There is increasing evidence that pregnancy alters the function of drug-metabolizing enzymes and drug transporters in a gestational-stage and tissue-specific manner. In vivo probe studies have shown that the activity of several hepatic cytochrome P450 enzymes, such as CYP2D6 and CYP3A4, is increased during pregnancy, whereas the activity of others, such as CYP1A2, is decreased. The activity of some renal transporters, including organic cation transporter and P-glycoprotein, also appears to be increased during pregnancy. Although much has been learned, significant gaps still exist in our understanding of the spectrum of drug metabolism and transport genes affected, gestational age–dependent changes in the activity of encoded drug metabolizing and transporting processes, and the mechanisms of pregnancy-induced alterations. In this issue of Drug Metabolism and Disposition, a series of articles is presented that address the predictability, mechanisms, and magnitude of changes in drug metabolism and transport processes during pregnancy. The articles highlight state-of-the-art approaches to studying mechanisms of changes in drug disposition during pregnancy, and illustrate the use and integration of data from in vitro models, animal studies, and human clinical studies. The findings presented show the complex inter-relationships between multiple regulators of drug metabolism and transport genes, such as estrogens, progesterone, and growth hormone, and their effects on enzyme and transporter expression in different tissues. The studies provide the impetus for a mechanism- and evidence-based approach to optimally managing drug therapies during pregnancy and improving treatment outcomes. PMID:23328895

Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse

PubMed Central

Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

2017-01-01

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS. PMID:28273875

Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse.

PubMed

Roca-Ho, Heleia; Riera, Marta; Palau, Vanesa; Pascual, Julio; Soler, Maria Jose

2017-03-05

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS.

In Healthy Young Men, a Short Exhaustive Exercise Alters the Oxidative Stress Only Slightly, Independent of the Actual Fitness

PubMed Central

Finkler, Maya; Hochman, Ayala; Pinchuk, Ilya; Lichtenberg, Dov

2016-01-01

The aim of the present study was to evaluate the apparent disagreement regarding the effect of a typical cycling progressive exercise, commonly used to assess VO2max, on the kinetics of ex vivo copper induced peroxidation of serum lipids. Thirty-two (32) healthy young men, aged 24–30 years, who do not smoke and do not take any food supplements, participated in the study. Blood was withdrawn from each participant at three time points (before the exercise and 5 minutes and one hour after exercise). Copper induced peroxidation of sera made of the blood samples was monitored by spectrophotometry. For comparison, we also assayed TBARS concentration and the activity of oxidation-related enzymes. The physical exercise resulted in a slight and reversible increase of TBARS and slight changes in the activities of the studied antioxidant enzymes and the lag preceding peroxidation did not change substantially. Most altered parameters returned to baseline level one hour after exercise. Notably, the exercise-induced changes in OS did not correlate with the physical fitness of the subjects, as evaluated in this study (VO2max = 30–60 mL/min/kg). We conclude that in healthy young fit men a short exhaustive exercise alters only slightly the OS, independent of the actual physical fitness. PMID:26989456

Alterations in carbohydrates and the protein metabolism of the harmful freshwater vector snail Lymnaea acuminata induced by the Euphorbia tirucalli latex extract.

PubMed

Tiwari, Sudhanshu; Singh, A

2005-11-01

To know the short- as well as long-term effect of aqueous latex extracts of Euphorbia tirucalli on carbohydrate and protein metabolism, the snail Lymnaea acuminata was exposed to sublethal doses of 0.37 and 0.55 mg/L for a 24-h and 0.20 and 0.31 mg/L for a 96-h exposure period. Significant (P<0.05) alterations in the glycogen, pyruvate, lactate, total protein, and free amino acid level, as well as in the activity of enzyme lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, protease, aspartate aminotransaminase, and alanine aminotransaminase were observed in the nervous, hepatopancreatic, and ovotestis tissues of the freshwater vector snail L. acuminata exposed to sublethal doses of E. tirucalli latex extract. The alterations in all biochemical parameters were significantly (P<0.05) time and dose dependent. After the 7th day of the withdrawal of treatment, there was significant (P<0.05) recovery in glycogen, pyruvate, lactate, total protein, and the free amino acid level and in the activity of the lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, protease, aspartate aminotransaminase and alanine aminotransaminase enzymes in all three of the studied tissues of the snail, which supports the view that the plant product is safe for use as a molluscicide for the control of harmful freshwater vector snails in the aquatic environment.

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.

Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

PubMed Central

Ueda, Norishi

2015-01-01

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

A semisynthetic strategy leads to alteration of the backbone amidate ligand in the NiSOD active site

DOE PAGES

Campeciño, Julius O.; Dudycz, Lech W.; Tumelty, David; ...

2015-07-01

Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategymore » that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ~1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N 2S 2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ~11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with g z < g x,y. 14N-hyperfine is observed on g z« less

RBC stearoyl-coA desaturase activity and the hepatic paradox in African descent women: the federal women's study

USDA-ARS?s Scientific Manuscript database

In women of African descent, low hepatic fat is paradoxically related to insulin resistance and cardiovascular disease. The reasons for the race/ethnic difference in the relationship between hepatic fat and IR are unclear but could be related to lower hepatic de novo lipogenesis due to altered enzym...

2,2",4,4"-TETRABROMODIPHENYL (PBDE 47) ALTERS THYROID FUNCTION IN THE RAT.

EPA Science Inventory

Two commercial PBDE mixtures, DE-71 and DE-79, cause dose-dependent depletion of serum T4 via induction of UGTs and increased CYP1A1 activity. This work characterized the effect of a major congener, PBDE-47, in DE-71 for effects on hepatic enzymes and thyroid hormones. Female 27...

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.

Biocatalytic induction of supramolecular order

NASA Astrophysics Data System (ADS)

Hirst, Andrew R.; Roy, Sangita; Arora, Meenakshi; Das, Apurba K.; Hodson, Nigel; Murray, Paul; Marshall, Stephen; Javid, Nadeem; Sefcik, Jan; Boekhoven, Job; van Esch, Jan H.; Santabarbara, Stefano; Hunt, Neil T.; Ulijn, Rein V.

2010-12-01

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure.

Base Excision Repair of Tandem Modifications in a Methylated CpG Dinucleotide*

PubMed Central

Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S.; Beard, William A.; Wilson, Samuel H.

2014-01-01

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3′-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5′-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide. PMID:24695738

Oxidative stress indices in gastroenteritis in dogs with canine parvoviral infection.

PubMed

Panda, Debasis; Patra, R C; Nandi, S; Swarup, D

2009-02-01

Gastroenteritis of viral origin has emerged as a major cause of morbidity and mortality in dogs during the last two decades. Amongst the viral etiologies responsible for gastroenteritis in dogs, canine parvovirus (CPV) is considered as the most pathogenic. The disease is characterized by hemorrhagic enteritis, bloody diarrhoea and myocarditis in young pups. The present study was carried out to examine alterations in oxidative stress indices in the erythrocytes from dogs suffering from gastroenteritis with or without canine parvoviral infection as confirmed by CPV-DNA amplification from faeces using specific primers for CPV-2 as well as CPV-2a and CPV-2b variants by polymerase chain reaction (PCR). The present investigation utilized clinical cases of dogs with signs of acute diarrhea (n=56), and 14 more apparently healthy dogs of similar age group. Erythrocytic oxidative stress indices such as lipid peroxides level and antioxidant enzymes like superoxide dismutase and catalase activity, and blood micro-mineral (iron, copper, cobalt and zinc) status were analyzed in each dog (n=70). The acute cases of gastroenteritis in dogs were associated with altered erythrocytic lipid peroxidation as evident by estimation of malonaldehyde (MDA) concentration. The activities of antioxidant enzymes catalase and superoxide dismutase, the first line of antioxidant defense against damaging effects of free radicals, were also altered. The alterations in oxidative stress indices were more pronounced in cases with involvement of canine parvovirus as compared to parvo-negative cases. Our results also revealed decreased blood zinc level in diarrhoea in dogs irrespective of involvement of canine parvovirus.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Altered lipid peroxidation markers are related to post-traumatic stress disorder (PTSD) and not trauma itself in earthquake survivors.

PubMed

Atli, Abdullah; Bulut, Mahmut; Bez, Yasin; Kaplan, İbrahim; Özdemir, Pınar Güzel; Uysal, Cem; Selçuk, Hilal; Sir, Aytekin

2016-06-01

The traumatic life events, including earthquakes, war, and interpersonal conflicts, cause a cascade of psychological and biological changes known as post-traumatic stress disorder (PTSD). Malondialdehyde (MDA) is a reliable marker of lipid peroxidation, and paraoxonase is a known antioxidant enzyme. The aims of this study were to investigate the relationship between earthquake trauma, PTSD effects on oxidative stress and the levels of serum paraoxonase 1 (PON1) enzyme activity, and levels of serum MDA. The study was carried out on three groups called: the PTSD group, the traumatized with earthquake exercise group, and healthy control group, which contained 32, 31, and 38 individuals, respectively. Serum MDA levels and PON1 enzyme activities from all participants were measured, and the results were compared across all groups. There were no significant differences between the PTSD patients and non-PTSD earthquake survivors in terms of the study variables. The mean PON1 enzyme activity from PTSD patients was significantly lower, while the mean MDA level was significantly higher than that of the healthy control group (p < 0.01 for both measurements). Similarly, earthquake survivors who did not develop PTSD showed higher MDA levels and lower PON1 activity when compared to healthy controls. However, the differences between these groups did not reach a statistically significant level. Increased MDA level and decreased PON1 activity measured in PTSD patients after earthquake and may suggest increased oxidative stress in these patients. The nonsignificant trends that are observed in lipid peroxidation markers of earthquake survivors may indicate higher impact of PTSD development on these markers than trauma itself. For example, PTSD diagnosis seems to add to the effect of trauma on serum MDA levels and PON1 enzyme activity. Thus, serum MDA levels and PON1 enzyme activity may serve as biochemical markers of PTSD diagnosis.

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

PubMed

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

2016-11-14

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity.

PubMed

Larrimore, Katherine E; Kazan, I Can; Kannan, Latha; Kendle, R Player; Jamal, Tameem; Barcus, Matthew; Bolia, Ashini; Brimijoin, Stephen; Zhan, Chang-Guo; Ozkan, S Banu; Mor, Tsafrir S

2017-09-05

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme's ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.

Transcriptome, antioxidant enzyme activity and histopathology analysis of hepatopancreas from the white shrimp Litopenaeus vannamei fed with aflatoxin B1(AFB1).

PubMed

Zhao, Wei; Wang, Lei; Liu, Mei; Jiang, Keyong; Wang, Mengqiang; Yang, Guang; Qi, Cancan; Wang, Baojie

2017-09-01

Aflatoxin produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing and storage. Aflatoxins cause severe health problems reducing the yield and profitability of shrimp cultures. We sought to understand the interaction between shrimp immunity and aflatoxin B1 (AFB1), analyzing transcriptome expression, antioxidant enzyme activity, and histological features of the hepatopancreas of shrimp fed with AFB1. From over 4 million high-quality reads, de novo unigene assembly produced 103,644 fully annotated genes. A total of 1024 genes were differentially expressed in shrimp fed with AFB1, being involved in functions, such as peroxidase metabolism, signal transduction, transcriptional control, apoptosis, proteolysis, endocytosis, and cell adhesion and cell junction. Upon AFB1 challenge, there were severe histological alterations in shrimp hepatopancreas. AFB1 challenge increased the activity of several antioxidant enzymes. Our data contribute to improve the current understanding of host-AFB1 interaction, providing an abundant source for identification of novel genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

PubMed Central

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

PubMed

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

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.

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

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain?

PubMed

Jha, Indrani; Bisht, Meena; Venkatesu, Pannuru

2016-06-30

The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01-0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.

The Effects of Space Flight on Some Liver Enzymes Concerned with Carbohydrate and Lipid Metabolism in Rats

NASA Technical Reports Server (NTRS)

Abraham, S.; Lin, C. Y.; Klein, H. P.; Volkmann, C.

1978-01-01

The activities of about 30 enzymes concerned with carbohydrate and lipid metabolism and the levels of glycogen and of individual fatty acids were measured in livers of rats ex- posed to prolonged space flight (18.5 days) aboard COSMOS 986 Biosatellite. When flight stationary, (FS) and flight centrifuged (FC) rats were compared at recovery (R(sub 0)), decrceases in the activities of glycogen phosphorylase, alpha glycerphosphate, acyl transferase, diglyceride acyl transferase, acconitase and Epsilon-phosphogluconate dehydrogenase were noted in the weightless group (FS). The significance of these findings was strengthened since all activities, showing alterations at R(sub 0), returned to normal 25 days post-flight. Differences were also seen in levels of two liver constituents. When glycogen and total fatty acids of the two groups of flight animals were determined, differences that could be attributed to reduced gravity were observed, the FS group at R(sub 0) contained, on the average, more than twice the amount of glycogen than did controls ad a remarkable shift in the ratio of palmitate to palmitoleate were noted. These metabolic alterations appear to be unique to the weightless condition. Our data justify the conclusion that centrifugation during space flight is equivalent to terrestrial gravity.

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

PubMed

Kumar, J K; Tabor, S; Richardson, C C

2001-09-14

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

Antioxidant enzymes expression in Pseudomonas aeruginosa exposed to UV-C radiation.

PubMed

Salma, Kloula Ben Ghorbal; Lobna, Maalej; Sana, Khefacha; Kalthoum, Chourabi; Imene, Ouzari; Abdelwaheb, Chatti

2016-07-01

It was well known that, UV-C irradiation increase considerably the reactive oxygen species (ROS) levels in eukaryotic and prokaryotic organisms. In the enzymatic ROS-scavenging pathways, superoxide dismutase (SOD), Catalase (CAT), and peroxidase (POX) were developed to deal with oxidative stress. In this study, we investigated the effects of UV-C radiations on antioxidant enzymes (catalase, superoxide dismutase, and peroxidases) expression in Pseudomonas aeruginosa. Catalase, superoxide dismutase, and peroxidases activities were determined spectrophotometrically. Isozymes of superoxide dismutase were revealed by native gel activity staining method. Lipid peroxidation was determined by measuring malondialdehyde formation. Our results showed that superoxide dismutase, catalase and peroxidase activities exhibited a gradual increase during the exposure time (30 min). However, the superoxide dismutase activity was maximized at 15 min. Native gel activity staining assays showed the presence of three superoxide dismutase isozymes. The iron-cofactored isoform activity was altered after exposure to UV-C stress. These finding suggest that catalase and peroxidase enzymes have the same importance toward UV-C rays at shorter and longer exposure times and this may confer additional protection to superoxide dismutase from damage caused by lipid peroxidation. Moreover, our data demonstrate the significant role of the antioxidant system in the resistance of this important human pathogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences.

PubMed

Kulikova, Vitalia V; Zakomirdina, Ludmila N; Dementieva, Irene S; Phillips, Robert S; Gollnick, Paul D; Demidkina, Tatyana V; Faleev, Nicolai G

2006-04-01

Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.

Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

PubMed Central

Rey, Pascal; Sanz-Barrio, Ruth; Innocenti, Gilles; Ksas, Brigitte; Courteille, Agathe; Rumeau, Dominique; Issakidis-Bourguet, Emmanuelle; Farran, Inmaculada

2013-01-01

Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast. PMID:24137166

Changes in skeletal muscle biochemistry and histology relative to fiber type in rats with heart failure.

PubMed

Delp, M D; Duan, C; Mattson, J P; Musch, T I

1997-10-01

One of the primary consequences of left ventricular dysfunction (LVD) after myocardial infarction is a decrement in exercise capacity. Several factors have been hypothesized to account for this decrement, including alterations in skeletal muscle metabolism and aerobic capacity. The purpose of this study was to determine whether LVD-induced alterations in skeletal muscle enzyme activities, fiber composition, and fiber size are 1) generalized in muscles or specific to muscles composed primarily of a given fiber type and 2) related to the severity of the LVD. Female Wistar rats were divided into three groups: sham-operated controls (n = 13) and rats with moderate (n = 10) and severe (n = 7) LVD. LVD was surgically induced by ligating the left main coronary artery and resulted in elevations (P < 0.05) in left ventricular end-diastolic pressure (sham, 5 +/- 1 mmHg; moderate LVD, 11 +/- 1 mmHg; severe LVD, 25 +/- 1 mmHg). Moderate LVD decreased the activities of phosphofructokinase (PFK) and citrate synthase in one muscle composed of type IIB fibers but did not modify fiber composition or size of any muscle studied. However, severe LVD diminished the activity of enzymes involved in terminal and beta-oxidation in muscles composed primarily of type I fibers, type IIA fibers, and type IIB fibers. In addition, severe LVD induced a reduction in the activity of PFK in type IIB muscle, a 10% reduction in the percentage of type IID/X fibers, and a corresponding increase in the portion of type IIB fibers. Atrophy of type I fibers, type IIA fibers, and/or type IIB fibers occurred in soleus and plantaris muscles of rats with severe LVD. These data indicate that rats with severe LVD after myocardial infarction exhibit 1) decrements in mitochondrial enzyme activities independent of muscle fiber composition, 2) a reduction in PFK activity in type IIB muscle, 3) transformation of type IID/X to type IIB fibers, and 4) atrophy of type I, IIA, and IIB fibers.

Changes in skeletal muscle biochemistry and histology relative to fiber type in rats with heart failure

NASA Technical Reports Server (NTRS)

Delp, M. D.; Duan, C.; Mattson, J. P.; Musch, T. I.

1997-01-01

One of the primary consequences of left ventricular dysfunction (LVD) after myocardial infarction is a decrement in exercise capacity. Several factors have been hypothesized to account for this decrement, including alterations in skeletal muscle metabolism and aerobic capacity. The purpose of this study was to determine whether LVD-induced alterations in skeletal muscle enzyme activities, fiber composition, and fiber size are 1) generalized in muscles or specific to muscles composed primarily of a given fiber type and 2) related to the severity of the LVD. Female Wistar rats were divided into three groups: sham-operated controls (n = 13) and rats with moderate (n = 10) and severe (n = 7) LVD. LVD was surgically induced by ligating the left main coronary artery and resulted in elevations (P < 0.05) in left ventricular end-diastolic pressure (sham, 5 +/- 1 mmHg; moderate LVD, 11 +/- 1 mmHg; severe LVD, 25 +/- 1 mmHg). Moderate LVD decreased the activities of phosphofructokinase (PFK) and citrate synthase in one muscle composed of type IIB fibers but did not modify fiber composition or size of any muscle studied. However, severe LVD diminished the activity of enzymes involved in terminal and beta-oxidation in muscles composed primarily of type I fibers, type IIA fibers, and type IIB fibers. In addition, severe LVD induced a reduction in the activity of PFK in type IIB muscle, a 10% reduction in the percentage of type IID/X fibers, and a corresponding increase in the portion of type IIB fibers. Atrophy of type I fibers, type IIA fibers, and/or type IIB fibers occurred in soleus and plantaris muscles of rats with severe LVD. These data indicate that rats with severe LVD after myocardial infarction exhibit 1) decrements in mitochondrial enzyme activities independent of muscle fiber composition, 2) a reduction in PFK activity in type IIB muscle, 3) transformation of type IID/X to type IIB fibers, and 4) atrophy of type I, IIA, and IIB fibers.

A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

PubMed

Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G; Elpeleg, Orly

2014-01-01

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

PubMed Central

Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G.; Elpeleg, Orly

2014-01-01

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred. PMID:25272002

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

PubMed

Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2008-11-01

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

Biochemical surface modification of Co-Cr-Mo.

PubMed

Puleo, D A

1996-01-01

Because of the limited mechanical properties of tissue substitutes formed by culturing cells on polymeric scaffolds, other approaches to tissue engineering must be explored for applications that require complete and immediate ability to bear weight, e.g. total joint replacements. Biochemical surface modification offers a way to partially regulate events at the bone-implant interface to obtain preferred tissue responses. Tresyl chloride, gamma-aminopropyltriethoxysilane (APS) and p-nitrophenyl chloroformate (p-NPC) immobilization schemes were used to couple a model enzyme, trypsin, on bulk samples of Co-Cr-Mo. For comparison, samples were simply adsorbed with protein. The three derivatization schemes resulted in different patterns and levels of activity. Tresyl chloride was not effective in immobilizing active enzyme on Co-Cr-Mo. Aqueous silanization with 12.5% APS resulted in optimal immobilized activity. Activity on samples derivatized with 0.65 mg p-NPC cm-2 was four to five times greater than that on samples simple adsorbed with enzyme or optimally derivatized with APS and was about eight times that on tresylated samples. This work demonstrates that, although different methods have different effectiveness, chemical derivatization can be used to alter the amount and/or stability of biomolecules immobilized on the surface of Co-Cr-Mo.

Soil Properties, Nutrient Dynamics, and Soil Enzyme Activities Associated with Garlic Stalk Decomposition under Various Conditions

PubMed Central

Han, Xu; Cheng, Zhihui; Meng, Huanwen

2012-01-01

The garlic stalk is a byproduct of garlic production and normally abandoned or burned, both of which cause environmental pollution. It is therefore appropriate to determine the conditions of efficient decomposition, and equally appropriate to determine the impact of this decomposition on soil properties. In this study, the soil properties, enzyme activities and nutrient dynamics associated with the decomposition of garlic stalk at different temperatures, concentrations and durations were investigated. Stalk decomposition significantly increased the values of soil pH and electrical conductivity. In addition, total nitrogen and organic carbon concentration were significantly increased by decomposing stalks at 40°C, with a 5∶100 ratio and for 10 or 60 days. The highest activities of sucrase, urease and alkaline phosphatase in soil were detected when stalk decomposition was performed at the lowest temperature (10°C), highest concentration (5∶100), and shortest duration (10 or 20 days). The evidence presented here suggests that garlic stalk decomposition improves the quality of soil by altering the value of soil pH and electrical conductivity and by changing nutrient dynamics and soil enzyme activity, compared to the soil decomposition without garlic stalks. PMID:23226411

Development and reduction of hypertension and oxidative stress among detergent industry workers.

PubMed

Boojar, Massod M A; Goodarzi, Faranak; Boojar, Manochehr M A

2004-12-01

Hypertension status and oxidative stress parameters were assessed in 291 workers (hypertensive workers were divided into three grades, non-equivalently) at two detergent production plants, one of which included enzymes in the detergent (n=138) and another which did not (n=153), and 45 control workers in another industry three times (at the time of employment, 7 yrs later at the time of installation of a filter system, and about 3 yrs later). Malondialdehyde (MDA) was measured by high-performance liquid chromatography, antioxidant enzymes and lipid status by ultraviolet-visible spectrophotometry, trace elements by atomic absorption spectroscopy, and blood pressure using an oscilometric device. Prior to filter system installation, enzyme-exposed workers had significantly higher MDA, antioxidant enzyme activities, and prevalence of hypertension, compared with controls. The filter system reduced airborne detergent and enzyme dusts, resulting in a decreased prevalence of hypertension and a significant improvement in workers' oxidative stress indicators. Alterations in antioxidant status may result from the cumulative effect of high levels of detergent and enzyme in airborne dust in the workplace.

Modification of erythrocyte membrane proteins, enzymes and transport mechanisms in chronic alcoholics: an in vivo and in vitro study.

PubMed

Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

2013-01-01

The aim of the study was to elucidate the molecular mechanisms underlying the alcohol perturbation leading to deleterious effects on erythrocyte membrane transport in chronic alcoholics. Membrane bound enzyme activities such as Na(+), K(+)-ATPase, Ca(2+),Mg(2+)-ATPase and acetylcholine esterase and membrane transport analysis by in vitro and erythrocyte membrane profile analysis in controls and chronic alcoholic red cells were analyzed. It was observed that decreased Na(+), K(+)-ATPase enzyme activity and increased activities of Ca(2+),Mg(2+)-ATPase and acetylcholine esterase in chronic alcoholics compared to controls. The in vitro studies of erythrocytes suggested that there is an increased uptake of glucose through chronic alcoholic red cells. However, glucose utilization by chronic alcoholic red cells was decreased. An increased sensitivity of ouabain for its binding site on Na(+), K(+)-ATPase in chronic alcoholic erythrocyte membrane was evident from this study. Though there appears to be an increased Na(+) influx in chronic alcoholic cells, the status of Na(+) transport is not altered much. However, ouabain caused slight disturbances in the transport of sodium, similar disturbances in the potassium transport resulting in much accumulation of potassium in red cells. It was concluded that chronic alcohol consumption modified certain membrane bound proteins, enzymes and transport mechanisms in chronic alcoholics.

Genetic determinants of drug responsiveness and drug interactions.

PubMed

Caraco, Y

1998-10-01

Six cytochrome P450 enzymes mediate the oxidative metabolism of most drugs in common use: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. These enzymes have selective substrate specificity, and their activity is characterized by marked interindividual variation. Some of these systems (CYP2C19, CYP2D6) are polymorphically distributed; thus, a subset of the population may be genetically deficient in enzyme activity. Phenotyping procedures designed to identify subjects with impaired metabolism who may be at increased risk for drug toxicity have been developed and validated. This has been supplemented in recent years by the availability of genetic analysis and the identification of specific alleles that are associated with altered (i.e., reduced, deficient, or increased) enzyme activity. The potential of genotyping to predict pharmacodynamics holds great promise for the future because it does not involve the administration of exogenous compound and is not confounded by drug therapy. Drug interactions caused by the inhibition or induction of oxidative drug metabolism may be of great clinical importance because they may result in drug toxicity or therapeutic failure. Further understanding of cytochrome P450 complexity may allow, through a combined in vitro-in vivo approach, the reliable prediction and possible prevention of deleterious drug interactions.

Mitochondrial Bioenergetics and Dysfunction in Failing Heart.

PubMed

Sheeran, Freya L; Pepe, Salvatore

2017-01-01

Energy insufficiency has been recognized as a key feature of systolic heart failure. Although mitochondria have long been known to sustain myocardial work energy supply, the capacity to therapeutically target mitochondrial bioenergetics dysfunction is hampered by a complex interplay of multiple perturbations that progressively compound causing myocardial failure and collapse. Compared to non-failing human donor hearts, activity rates of complexes I and IV, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase, Nnt) and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase are markedly decreased in end-stage heart failure. Diminished REDOX capacity with lower total glutathione and coenzyme Q 10 levels are also a feature of chronic left ventricular failure. Decreased enzyme activities in part relate to abundant and highly specific oxidative, nitrosylative, and hyperacetylation modifications. In this brief review we highlight that energy deficiency in end-stage failing human left ventricle predominantly involves concomitantly impaired activities of key electron transport chain and Krebs cycle enzymes rather than altered expression of respective genes or proteins. Augmented oxidative modification of these enzyme subunit structures, and the formation of highly reactive secondary metabolites, implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, which contribute further to progressive decreases in bioenergetic capacity and contractile function in human heart failure.

Short-Term and Sub-Chronic Dietary Exposure to Aspalathin-Enriched Green Rooibos (Aspalathus linearis) Extract Affects Rat Liver Function and Antioxidant Status.

PubMed

van der Merwe, Johanna Debora; de Beer, Dalene; Joubert, Elizabeth; Gelderblom, Wentzel C A

2015-12-18

An aspalathin-enriched green rooibos (Aspalathus linearis) extract (GRE) was fed to male Fischer rats in two independent studies for 28 and 90 days. The average dietary total polyphenol (TP) intake was 756 and 627 mg Gallic acid equivalents (GAE)/kg body weight (bw)/day over 28 and 90 days, respectively, equaling human equivalent doses (HEDs) of 123 and 102 GAE mg/kg bw/day. Aspalathin intake of 295 mg/kg bw/day represents a HED of 48 mg/kg bw/day (90 day study). Consumption of GRE increased feed intake significantly (p < 0.05) compared to the control after 90 days, but no effect on body and organ weight parameters was observed. GRE significantly (p < 0.05) reduced serum total cholesterol and iron levels, whilst significantly (p < 0.05) increasing alkaline phosphatase enzyme activity after 90 days. Endogenous antioxidant enzyme activity in the liver, i.e., catalase and superoxide dismutase activity, was not adversely affected. Glutathione reductase activity significantly (p < 0.05) increased after 28 days, while glutathione (GSH) content was decreased after 90 days, suggesting an altered glutathione redox cycle. Quantitative Real Time polymerase chain reaction (PCR) analysis showed altered expression of certain antioxidant defense and oxidative stress related genes, indicative, among others, of an underlying oxidative stress related to changes in the GSH redox pathway and possible biliary dysfunction.

The interactive effects of mercury and selenium on metabolic profiles, gene expression and antioxidant enzymes in halophyte Suaeda salsa.

PubMed

Liu, Xiaoli; Lai, Yongkai; Sun, Hushan; Wang, Yiyan; Zou, Ning

2016-04-01

Suaeda salsa is the pioneer halophyte in the Yellow River Delta and was consumed as a popular vegetable. Mercury has become a highly risky contaminant in the sediment of intertidal zones of the Yellow River Delta. In this work, we investigated the interactive effects of mercury and selenium in S. salsa on the basis of metabolic profiling, antioxidant enzyme activities and gene expression quantification. Our results showed that mercury exposure (20 μg L(-1)) inhibited plant growth of S. salsa and induced significant metabolic responses and altered expression levels of INPS, CMO, and MDH in S. salsa samples, together with the increased activities of antioxidant enzymes including SOD and POD. Overall, these results indicated osmotic and oxidative stresses, disturbed protein degradation and energy metabolism change in S. salsa after mercury exposures. Additionally, the addition of selenium could induce both antagonistic and synergistic effects including alleviating protein degradation and aggravating osmotic stress caused by mercury. © 2014 Wiley Periodicals, Inc.

Royal jelly attenuates azathioprine induced toxicity in rats.

PubMed

Ahmed, Walaa M S; Khalaf, A A; Moselhy, Walaa A; Safwat, Ghada M

2014-01-01

In the present study, we investigated the potential protective effects of royal jelly against azathioprine-induced toxicity in rat. Intraperitoneal administration of azathioprine (50 mg/kgB.W.) induced a significant decrease in RBCs count, Hb concentration, PCV%, WBCs count, differential count and platelet count, hepatic antioxidant enzymes (reduced glutathione and glutathione s-transferase) and increase of serum transaminases (alanine aminotransferase and aspartate aminotransferase enzymes) activities, alkaline phosphatase and malondialdehyde formation. Azathioprine induced hepatotoxicity was reflected by marked pathological changes in the liver. Oral administration of royal jelly (200 mg/kgB.W.) was efficient in counteracting azathioprine toxicity whereas it altered the anemic condition, leucopenia and thrombocytopenia induced by azathioprine. Furthermore, royal jelly exerted significant protection against liver damage induced by azathioprine through reduction of the elevated activities of serum hepatic enzymes. Moreover, royal jelly blocked azathioprine-induced lipid peroxidation through decreasing the malondialdehyde formation. In conclusion, royal jelly possesses a capability to attenuate azathioprine-induced toxicity. Copyright © 2013 Elsevier B.V. All rights reserved.

Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

PubMed Central

Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R.

2016-01-01

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients. PMID:27725636

Differences in response of glucuronide and glutathione conjugating enzymes to aflatoxin B/sub 1/ and N-acetylaminofluorene in underfed rats

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

Rajpurohit, R.; Krishnaswamy, K.

Changes in the hepatic drug/xenobiotic-metabolizing enzymes in underfed rats exposed to aflatoxin B/sub 1/ and N-acetylaminofluorene were investigated. Neither carcinogen, fed at the level of 10 ..mu..g and 0.667 mg per 100 g body weight, respectively, over a period of 3 wk, had any significant influence on cytochrome P-450 and aryl hydrocarbon hydroxylase in the undernourished rats. Significantly low activities of UDP-glucuronyltransferase and glutathione S-transferase were observed in food-restricted animals fed on aflatoxin B/sub 1/. N-acetylaminofluorene, on the other hand stimulated both the enzyme activities in the underfed group, to as much observed in the respective well-fed treated group. UDP-Glucuronyltransferasemore » and glutathione S-transferase in undernutrition seem to respond differently to aflatoxin B/sub 1/ and N-acetylaminofluorene. Further studies are needed to assess the possible consequences of such alterations.« less

Glyphosate degradation in glyphosate-resistant and -susceptible crops and weeds.

PubMed

Duke, Stephen O

2011-06-08

High levels of aminomethylphosphonic acid (AMPA), the main glyphosate metabolite, have been found in glyphosate-treated, glyphosate-resistant (GR) soybean, apparently due to plant glyphosate oxidoreductase (GOX)-like activity. AMPA is mildly phytotoxic, and under some conditions the AMPA accumulating in GR soybean correlates with glyphosate-caused phytotoxicity. A bacterial GOX is used in GR canola, and an altered bacterial glyphosate N-acetyltransferase is planned for a new generation of GR crops. In some weed species, glyphosate degradation could contribute to natural resistance. Neither an isolated plant GOX enzyme nor a gene for it has yet been reported in plants. Gene mutation or amplification of plant genes for GOX-like enzyme activity or horizontal transfer of microbial genes from glyphosate-degrading enzymes could produce GR weeds. Yet, there is no evidence that metabolic degradation plays a significant role in evolved resistance to glyphosate. This is unexpected, considering the extreme selection pressure for evolution of glyphosate resistance in weeds and the difficulty in plants of evolving glyphosate resistance via other mechanisms.

The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

PubMed Central

Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O.; Tang, Yi; Houk, K. N.

2014-01-01

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF. PMID:24727900

Age characteristics of changes in invertase activity of the mucous membrane of the small intestine

NASA Technical Reports Server (NTRS)

Rakhimov, K. R.; Aleksandrova, N. V.

1980-01-01

Rats of varying ages were subjected to stress from heat, cold, and hydrocortisone injection. Invertase activity in homogenates of small intestine mucous membranes was studied following sacrifice. Invertase activity was low in young animals, but increased sharply in 30 day old ones, remaining at a relatively constant level until old age. The study concludes that the stress hormone (corticosteroids, etc.) levels in the blood, which affects the formation of enteric enzyme levels and activities, and that age related peculiarities in invertase activity are a consequence of altered hormone status and epitheliocyte sensitivity.

Effect of parasitic infection on dopamine biosynthesis in dopaminergic cells

PubMed Central

Martin, H.L.; Alsaady, I.; Howell, G.; Prandovszky, E.; Peers, C.; Robinson, P.; McConkey, G.A.

2015-01-01

Infection by the neurotropic agent Toxoplasma gondii alters rodent behavior and can result in neuropsychiatric symptoms in humans. Little is understood regarding the effects of infection on host neural processes but alterations to dopaminergic neurotransmission are implicated. We have previously reported elevated levels of dopamine (DA) in infected dopaminergic cells however the involvement of the host enzymes and fate of the produced DA were not defined. In order to clarify the effects of infection on host DA biosynthetic enzymes and DA packaging we examined enzyme levels and activity and DA accumulation and release in T. gondii-infected neurosecretory cells. Although the levels of the host tyrosine hydroxylase (TH) and DOPA decarboxylase and AADC (DDC) did not change significantly in infected cultures, DDC was found within the parasitophorous vacuole (PV), the vacuolar compartment where the parasites reside, as well as in the host cytosol in infected dopaminergic cells. Strikingly, DDC was found within the intracellular parasite cysts in infected brain tissue. This finding could provide some explanation for observations of DA within tissue cysts in infected brain as a parasite-encoded enzyme with TH activity was also localized within tissue cysts. In contrast, cellular DA packaging appeared unchanged in single-cell microamperometry experiments and only a fraction of the increased DA was accessible to high potassium-induced release. This study provides some understanding of how this parasite produces elevated DA within dopaminergic cells without the toxic ramifications of free cytosolic DA. The mechanism for synthesis and packaging of DA by T. gondii-infected dopaminergic cells may have important implications for the effects of chronic T. gondii infection on humans and animals. PMID:26297895

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

PubMed

Hansdottir, Sif; Monick, Martha M; Hinde, Sara L; Lovan, Nina; Look, Dwight C; Hunninghake, Gary W

2008-11-15

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Effects of chronic caloric restriction on kidney and heart redox status and antioxidant enzyme activities in Wistar rats

PubMed Central

Dutra, Márcio Ferreira; Bristot, Ivi Juliana; Batassini, Cristiane; Cunha, Núbia Broetto; Vizuete, Adriana Fernanda Kuckartz; de Souza, Daniela Fraga; Moreira, José Cláudio Fonseca; Gonçalves, Carlos-Alberto

2012-01-01

Caloric restriction (CR) has been associated with health benefits and these effects have been attributed, in part, to modulation of oxidative status by CR; however, data are still controversial. Here, we investigate the effects of seventeen weeks of chronic CR on parameters of oxidative damage/modification of proteins and on antioxidant enzyme activities in cardiac and kidney tissues. Our results demonstrate that CR induced an increase in protein carbonylation in the heart without changing the content of sulfhydryl groups or the activities of superoxide dismutase and catalase (CAT). Moreover, CR caused an increase in CAT activity in kidney, without changing other parameters. Protein carbonylation has been associated with oxidative damage and functional impairment; however, we cannot exclude the possibility that, under our conditions, this alteration indicates a different functional meaning in the heart tissue. In addition, we reinforce the idea that CR can increase CAT activity in the kidney. [BMB Reports 2012; 45(11): 671-676] PMID:23187008

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

PubMed

Wood, Chris M; Kajimura, Makiko; Mommsen, Thomas P; Walsh, Patrick J

2008-01-01

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

PubMed

Cárcamo, Juan Guillermo; Aguilar, Marcelo N; Carreño, Constanza F; Vera, Tamara; Arias-Darraz, Luis; Figueroa, Jaime E; Romero, Alex P; Alvarez, Marco; Yañez, Alejandro J

2017-01-01

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution of allosteric regulation in chorismate mutases from early plants

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

Kroll, Kourtney; Holland, Cynthia K.; Starks, Courtney M.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plantmore » chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.« less

Systemic mast cell activation disease: the role of molecular genetic alterations in pathogenesis, heritability and diagnostics

PubMed Central

Haenisch, Britta; Nöthen, Markus M; Molderings, Gerhard J

2012-01-01

Despite increasing understanding of its pathophysiology, the aetiology of systemic mast cell activation disease (MCAD) remains largely unknown. Research has shown that somatic mutations in kinases are necessary for the establishment of a clonal mast cell population, in particular mutations in the tyrosine kinase Kit and in enzymes and receptors with crucial involvement in the regulation of mast cell activity. However, other, as yet undetermined, abnormalities are necessary for the manifestation of clinical disease. The present article reviews molecular genetic research into the identification of disease-associated genes and their mutational alterations. The authors also present novel data on familial systemic MCAD and review the associated literature. Finally, the importance of understanding the molecular basis of inherited mutations in terms of diagnostics and therapy is emphasized. PMID:22957768

Effects of 200 Gy 60Co-γ Radiation on the Regulation of Antioxidant Enzymes, Hsp70 Genes, and Serum Molecules of Plutella xylostella (Linnaeus).

PubMed

Li, Xiaoxue; Luo, Lingyan; Karthi, Sengodan; Zhang, Ke; Luo, Jianjun; Hu, Qiongbo; Weng, Qunfang

2018-04-26

The diamondback moth, Plutella xylostella (Linnaeus), is one of the notorious pests causing substantial loses to many cruciferous vegetables across the nations. The effects of 60 Co-γ radiation on physiology of P. xylostella were investigated and the results displayed that 200 Gy irradiation significantly alters the antioxidant enzyme regulation in six-day-old male pupae of P. xylostella . First, in our research, we detected Oxidase system and stress response mechanism of irradiated pupae, the results displayed that 200 Gy irradiation significantly alters the antioxidant enzyme regulation in six-day-old male pupae of P. xylostella . The levels of superoxide dismutase (SOD) and catalase (CAT) were increased significantly in contrast the level of peroxidase (POD) and glutathione S-transferase (GST) were decreased in 12⁻24 h post-treatment. The heat shock proteins (Hsps) gene expression level was significant increasing, maximum > 2-folds upregulation of genes were observed in peak. However, they also had a trend of gradual recovery with development. Second, we detected the testis lactate dehydrogenase (LDH) and acid phosphatase (ACP) activity found that in male adults testis they increased significantly than control during its development. Thus the present research investigation highlights that the 60 Co-γ radiation treatments alters the physiological development of diamondback moth. The results showed that 200 Gy dosage resulted in stress damage to the body and reproductive system of the diamondback moth.

Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio).

PubMed

Anichtchik, Oleg; Sallinen, Ville; Peitsaro, Nina; Panula, Pertti

2006-10-10

Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). We have reported earlier MPTP-related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO-A and MAO-B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform-specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain.

Caffeic acid and quercetin protect erythrocytes against the oxidative stress and the genotoxic effects of lambda-cyhalothrin in vitro.

PubMed

Abdallah, Fatma Ben; Fetoui, H; Fakhfakh, F; Keskes, L

2012-01-01

Lambda-cyhalothrin (LTC) is a synthetic pyrethroid with a broad spectrum of insecticidal and acaricidal activities used to control wide range of insect pests in a variety of applications. The aim of this study was to examine (i) the potency of LTC to induce oxidative stress response in rat erythrocytes in vitro and (ii) the role of caffeic acid (20 μM) and/or quercetin (10 μM) in preventing the cytotoxic effects. Erythrocytes were divided into four portions. The erythrocytes of the first portion were incubated for 4 h at 37°C with different concentrations (0, 50 and 100 μM) of LTC. The others portions were pretreated with caffeic acid and/or quercetin for 30 min prior to LTC incubation. Lipid peroxidation, protein oxidation, antioxidant enzyme activities and DNA damage were examined. LTC at different concentrations causes increased levels of lipid peroxidation, protein oxidation, DNA damage and decreased antioxidant enzyme activities. Combined caffeic acid and quercetin pretreatments significantly reduced the levels of lipid peroxidation markers, that is thiobarbituric acid reactive substance (TBARS), protein carbonyls (PCO) and decreased DNA damage in LTC portion. Further, combined caffeic acid and quercetin pretreatment maintain antioxidant enzyme activities and glutathione content near to normal values. These results suggest that LTC exerts its toxic effect by increasing lipid peroxidation, altering the antioxidant enzyme activities and DNA damage. Caffeic acid and quercetin pretreatments prevent the toxic effects of LTC, suggesting their role as a potential antioxidant.

Triacylglycerol mimetics regulate membrane interactions of glycogen branching enzyme: implications for therapy.

PubMed

Alvarez, Rafael; Casas, Jesús; López, David J; Ibarguren, Maitane; Suari-Rivera, Ariadna; Terés, Silvia; Guardiola-Serrano, Francisca; Lossos, Alexander; Busquets, Xavier; Kakhlon, Or; Escribá, Pablo V

2017-08-01

Adult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca 2+ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Effects of pyridostigmine and cholinolytics on cholinesterase and acetylcholine in Soman poisoned rats.

PubMed

Stitcher, D L; Harris, L W; Heyl, W C; Alter, S C

1978-01-01

Soman reduced blood and brain cholinesterase (ChE) activity to less than 15% and increased cerebral acetylcholine (ACh) levels to 137.4% of control. When pyridostigmine (P) was used as a prophylactic adjunct, it reduced blood ChE activity to 31.6% of control, failed to significantly alter brain ChE activity, and protected more than 70% of the blood (but not brain enzyme) from phosphonylation by soman. Benactyzine (B) was more effective than atropine (A) in reducing cerebral ACh concentrations, while a combination of the two was more effective than either alone. A prophylaxis of P + A + B was effective in controlling ACh levels in rats poisoned with one LD50 dose of Soman. Since P did not diminish the effects of the cholinolytics on cerebral ACh, this (together with the enzyme data) suggests that the two cholinolytics alone provided the central protection.

Effects of sulfate deposition on pore water dissolved organic carbon, nutrients, and microbial enzyme activities in a northern peatland

Treesearch

L.R. Seifert-Monson; B.H. Hill; R.K. Kolka; T.M. Jicha; L.L. Lehto; C.M. Elonen

2014-01-01

Export of dissolved organic carbon from lakes and streams has increased throughout Europe and North America over the past several decades. One possible cause is altered deposition chemistry; specifically, decreasing sulfate inputs leading to changes in ionic strength and dissolved organic carbon solubility. To further investigate the relationship between deposition...

Interactive plant functional group and water table effects on decomposition and extracellular enzyme activity in Sphagnum peatlands

Treesearch

Magdalena M. Wiedermann; Evan S. Kane; Lynette R. Potvin; Erik A. Lilleskov

2017-01-01

Peatland decomposition may be altered by hydrology and plant functional groups (PFGs), but exactly how the latter influences decomposition is unclear, as are potential interactions of these factors.We used a factorial mesocosm experiment with intact 1 m3 peat monoliths to explore how PFGs (sedges vs Ericaceae) and water table level individually...

Female mice lacking p47phox have altered adipose tissue gene expression and are protected against high fat-induced obesity

USDA-ARS?s Scientific Manuscript database

Oxidative stress in the fat and the liver has been linked to the development of obesity and the metabolic syndrome. However, the molecular origin of reactive oxygen species and the role of these in obesity remain areas of active investigation. The NADPH oxidases (NOX) enzymes are a major source of ...

Impact of maternal steroids during pregnancy.

PubMed

Reynolds, Rebecca M

2016-12-01

Increased fetal exposure to glucocorticoids is a key mechanism thought to underlie the early life programming of later life disease. There is substantial experimental data in animal models in support of this hypothesis. Emerging evidence suggests glucocorticoid programming may also occur in humans with some studies now linking maternal endogenous cortisol levels with size at birth and gestation at delivery. The dramatic changes to the maternal hypothalamic-pituitary-adrenal axis during pregnancy mean that large-scale studies in humans are challenging to conduct. One of the key regulators of fetal glucocorticoid exposure is the activity of placental "barrier" enzyme 11β-hydroxysteroid dehydrogenase type 2 (HSD2) which converts active cortisol to inactive cortisone. In animal models, this enzyme is down-regulated by various influences including maternal malnutrition, inflammation or stress but it is not known whether this is a major factor in regulation of human fetal glucocorticoid exposure. More studies are needed to understand the mechanisms whereby altered fetal glucocorticoid exposure may alter fetal growth trajectories and whether changes in the maternal hypothalamic-pituitary-adrenal axis in pregnancy could be suitable as a biomarker to identify those pregnancies most at risk. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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.

Maternal obesity alters feto-placental Cytochrome P4501A1 activity

PubMed Central

DuBois, Barent N.; O’Tierney, Perrie; Pearson, Jacob; Friedman, Jacob E.; Thornburg, Kent; Cherala, Ganesh

2012-01-01

Cytochrome P4501A1 (CYP1A1), an important drug metabolizing enzyme, is expressed in human placenta throughout gestation as well as in fetal liver. Obesity, a chronic inflammatory condition, is known to alter CYP enzyme expression in non-placental tissues. In the present study, we test the hypothesis that maternal obesity alters the distribution of CYP1A1 activity in feto-placental unit. Placentas were collected from non-obese (BMI<30) and obese (BMI>30) women at term. Livers were collected from gestation day 130 fetuses of non-human primates fed either control diet or high-fat diet (HFD). Cytosol and microsomes were collected using differential centrifugation, and incubated with 7-Ethoxyresorufin. The CYP1A1 specific activity (pmoles of resorufin formed/min/mg of protein) was measured at excitation/emission wavelength of 530/590nm. Placentas of obese women had significantly reduced microsomal CYP1A1 activity compared to non-obese women (0.046 vs. 0.082; p<0.05); however no such effect was observed on cytosolic activity. Similarly, fetal liver from HFD fed mothers had significantly reduced microsomal CYP1A1 activity (0.44±0.04 vs. 0.20±0.10; p<0.05), with no significant difference in cytosolic CYP1A1 activity (control, 1.23±0.20; HFD, 0.80±0.40). Interestingly, multiple linear regression analyses of placental efficiency indicates cytosolic CYP1A1 activity is a main effect (5.67±2.32 (β±SEM); p=0.022) along with BMI (−0.57±0.26; p=0.037), fetal gender (1.07±0.26; p<0.001), and maternal age (0.07±0.03; p=0.011). In summary, while maternal obesity affects microsomal CYP1A1 activity alone, cytosolic activity along with maternal BMI is an important determinant of placental efficiency. Together, these data suggest that maternal lifestyle could have a significant impact on CYP1A1 activity, and hints at a possible role for CYP1A1 in feto-placental growth and thereby well-being of fetus. PMID:23046808

Exercise Training positively modulates the Ectonucleotidase Enzymes in Lymphocytes of Metabolic Syndrome Patients.

PubMed

Martins, C C; Bagatini, M D; Cardoso, A M; Zanini, D; Abdalla, F H; Baldissarelli, J; Dalenogare, D P; Dos Santos, D L; Schetinger, M R C; Morsch, V M M

2016-11-01

In this study, we investigated the cardiovascular risk factors as well as ectonucleotidase activities in lymphocytes of metabolic syndrome (MetS) patients before and after an exercise intervention. 20 MetS patients, who performed regular concurrent exercise training for 30 weeks, 3 times/week, were studied. Anthropometric, biochemical, inflammatory and hepatic parameters and hydrolysis of adenine nucleotides and nucleoside in lymphocytes were collected from patients before and after 15 and 30 weeks of the exercise intervention as well as from participants of the control group. An increase in the hydrolysis of ATP and ADP, and a decrease in adenosine deamination in lymphocytes of MetS patients before the exercise intervention were observed (P<0.001). However, these alterations were reversed by exercise training after 30 weeks of intervention. Additionally, exercise training reduced the inflammatory and hepatic markers to baseline levels after 30 weeks of exercise. Our results clearly indicated alteration in ectonucleotidase enzymes in lymphocytes in the MetS, whereas regular exercise training had a protective effect on the enzymatic alterations and on inflammatory and hepatic parameters, especially if it is performed regularly and for a long period. © Georg Thieme Verlag KG Stuttgart · New York.

Flap Conformations in HIV-1 Protease are Altered by Mutations

NASA Astrophysics Data System (ADS)

Fanucci, Gail; Blackburn, Mandy; Veloro, Angelo; Galiano, Luis; Fangu, Ding; Simmerling, Carlos

2009-03-01

HIV-1 protease (PR) is an enzyme that is a major drug target in the treatment of AIDS. Although the structure and function of HIV-1 PR have been studied for over 20 years, questions remain regarding the conformations and dynamics of the β-hairpin turns (flaps) that cover the active site cavity. Distance measurements with pulsed EPR spectroscopy of spin labeled constructs of HIV-1 PR have been used to characterize the flap conformations in the apo and inhibitor bound states. From the most probably distances and the breadth of the distance distribution profiles from analysis of the EPR data, insights regarding the flap conformations and flexibility are gained. The EPR results clearly show how drug pressure selected mutations alter the average conformation of the flaps and the degree of opening of the flaps. Molecular dynamics simulations successfully regenerate the experimentally determined distance distribution profiles, and more importantly, provide structural models for full interpretation of the EPR results. By combining experiment and theory to understand the role that altered flap flexibility/conformations play in the mechanism of drug resistance, key insights are gained toward the rational development of new inhibitors of this important enzyme.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alters Mitochondrial Membrane Lipids

PubMed Central

Sandra, Ferry; Esposti, Mauro Degli; Ndebele, Kenneth; Gona, Philimon; Knight, David; Rosenquist, Magnus; Khosravi-Far, Roya

2010-01-01

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to have selective antitumor activity. TRAIL induces ubiquitous pathways of cell death in which caspase activation is mediated either directly or via the release of apoptogenic factors from mitochondria; however, the precise components of the mitochondrial signaling pathway have not been well defined. Notably, mitochondria constitute an important target in overcoming resistance to TRAIL in many types of tumors. Bid is considered to be fundamental in engaging mitochondria during death receptor–mediated apoptosis, but this action is dependent on mitochondrial lipids. Here, we report that TRAIL signaling induces an alteration in mitochondrial membrane lipids, particularly cardiolipin. This occurs independently of caspase activation and primes mitochondrial membranes to the proapoptotic action of Bid. We unveil a link between TRAIL signaling and alteration of membrane lipid homeostasis that occurs in parallel to apical caspase activation but does not take over the mode of cell death because of the concurrent activation of caspase-8. In particular, TRAIL-induced alteration of mitochondrial lipids follows an imbalance in the cellular homeostasis of phosphatidylcholine, which results in an elevation in diacylglycerol (DAG). Elevated DAG in turn activates the δ isoform of phospholipid-dependent serine/threonine protein kinase C, which then accelerates the cleavage of caspase-8. We also show that preservation of phosphatidylcholine homeostasis by inhibition of lipid-degrading enzymes almost completely impedes the activation of pro-caspase-9 while scarcely changing the activation of caspase-8. PMID:16166305

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

PubMed Central

Sauer, Aisha V.; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L.; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S.; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D.; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D’Adamo, Patrizia; Aiuti, Alessandro

2017-01-01

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency. PMID:28074903

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients.

PubMed

Sauer, Aisha V; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D'Adamo, Patrizia; Aiuti, Alessandro

2017-01-11

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

Characterization of impaired processing of neuropeptides in the brains of endoprotease knockout mice.

PubMed

Beinfeld, Margery C

2011-01-01

With the development of mice in which individual proteolytic enzymes have been inactivated, it has been of great interest to see how loss of these enzymes alters the processing of neuropeptides. In the course of studying changes in the peptide cholecystokinin (CCK) and other neuropeptides in several of these knockout mice, it has become clear that neuropeptide processing is complex and regionally specific. The enzyme responsible for processing in one part of the brain may not be involved in other parts of the brain. It is essential to do a detailed dissection of the brain and analyze peptide levels in many brain regions to fully understand the role of the enzymes. Because loss of these proteases may trigger compensatory mechanisms which involve expression of the neuropeptides being studied or other proteases or accessory proteins, it is also important to examine how loss of an enzyme alters expression of the neuropeptides being studied as well as other proteins thought to be involved in neuropeptide processing. By determining how loss of an enzyme alters the molecular form(s) of the peptide that are made, additional mechanistic information can be obtained. This review will describe established methods to achieve these research goals.

Antioxidant Properties and Cardioprotective Mechanism of Malaysian Propolis in Rats

PubMed Central

Ahmed, Romana; Hossen, Md. Sakib; Ahmmed, Istiyak; Rumpa, Nur-E-Noushin; Sulaiman, Siti Amrah

2017-01-01

Propolis contains high concentrations of polyphenols, flavonoids, tannins, ascorbic acid, and reducing sugars and proteins. Malaysian Propolis (MP) has been reported to exhibit high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and ferric reducing antioxidant power (FRAP) values. Herein, we report the antioxidant properties and cardioprotective properties of MP in isoproterenol- (ISO-) induced myocardial infarction in rats. Male Wistar rats (n = 32) were pretreated orally with an ethanol extract of MP (100 mg/kg/day) for 30 consecutive days. Subcutaneous injection of ISO (85 mg/kg in saline) for two consecutive days caused a significant increase in serum cardiac marker enzymes and cardiac troponin I levels and altered serum lipid profiles. In addition significantly increased lipid peroxides and decreased activities of cellular antioxidant defense enzymes were observed in the myocardium. However, pretreatment of ischemic rats with MP ameliorated the biochemical parameters, indicating the protective effect of MP against ISO-induced ischemia in rats. Histopathological findings obtained for the myocardium further confirmed the biochemical findings. It is concluded that MP exhibits cardioprotective activity against ISO-induced oxidative stress through its direct cytotoxic radical-scavenging activities. It is also plausible that MP contributed to endogenous antioxidant enzyme activity via inhibition of lipid peroxidation. PMID:28261310

Exogenous proline mediates alleviation of cadmium stress by promoting photosynthetic activity, water status and antioxidative enzymes activities of young date palm (Phoenix dactylifera L.).

PubMed

Zouari, M; Ben Ahmed, Ch; Zorrig, W; Elloumi, N; Rabhi, M; Delmail, D; Ben Rouina, B; Labrousse, P; Ben Abdallah, F

2016-06-01

The ability of exogenous compatible solutes, such as proline, to counteract cadmium (Cd) inhibitory effects in young date palm plants (Phoenix dactylifera L. cv Deglet Nour) was investigated. Two-year-old date palm plants were subjected for five months at different Cd stress levels (0, 10 and 30 mg CdCl2 kg(-1) soil) whether supplied or not with exogenous proline (20mM) added through the irrigation water. Different levels of Cd stress altered plant growth, gas exchanges and chlorophyll content as well as water status, but at different extent among them. In contrast, an increase of antioxidant enzymes activities of Cd-treated plants in association with high amounts of proline content, hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and electrolyte leakage (EL) were observed. Interestingly, exogenous proline mitigated the adverse effects of Cd on young date palm. Indeed, it alleviated the oxidative damage induced by Cd accumulation and established better levels of plant growth, water status and photosynthetic activity. Moreover, proline-treated plants showed high antioxidant enzymes activities (superoxide dismutase, catalase and glutathione peroxydase) in roots and leaves as compared to Cd-treated plants. Copyright © 2016 Elsevier Inc. All rights reserved.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

PubMed Central

Zhang, Jun; Preda, Dorin V.; Vasquez, Kristine O.; Morin, Jeff; Delaney, Jeannine; Bao, Bagna; Percival, M. David; Xu, Daigen; McKay, Dan; Klimas, Michael; Bednar, Bohumil; Sur, Cyrille; Gao, David Z.; Madden, Karen; Yared, Wael; Rajopadhye, Milind

2012-01-01

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments. PMID:22674025

Kinetic and biophysical investigation of the inhibitory effect of caffeine on human salivary aldehyde dehydrogenase: Implications in oral health and chemotherapy

NASA Astrophysics Data System (ADS)

Laskar, Amaj Ahmed; Alam, Md Fazle; Ahmad, Mohammad; Younus, Hina

2018-04-01

Human salivary aldehyde dehydrogenase (hsALDH) is primarily a class 3 ALDH (ALDH3A1), and is an important antioxidant enzyme present in the saliva which maintains healthy oral cavity. It detoxifies toxic aldehydes into non-toxic carboxylic acids in the oral cavity. Reduced level of hsALDH activity is a risk factor for oral cancer development. It is involved in the resistance of certain chemotherapeutic drugs. Coffee has been reported to affect the activity of salivary ALDH. In this study, the effect of caffeine on the activity (dehydrogenase and esterase) of hsALDH was investigated. The binding of caffeine to hsALDH was studied using different biophysical methods and molecular docking analysis. Caffeine was found to inhibit both crude and purified hsALDH. The Km increased and the Vmax decreased showing a mixed type of inhibition. Caffeine decreased the nucleophilicity of the catalytic cysteine residue. It binds to the active site of ALDH3A1 by forming a complex through non-covalent interactions with some highly conserved amino acid residues. It partially alters the secondary structure of the enzyme. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing substrate binding affinity and the catalytic efficiency of the enzyme. The study indicates that oral intake of caffeine may have a harmful effect on the oral health and may increase the risk of carcinogenesis through the inhibition of this important enzyme. Further, the inactivation of oxazaphosphorine based chemotherapeutic drugs by ALDH3A1 may be prevented by using caffeine as an adjuvant during medication which is expected to increase the sensitivity of these drugs through its inhibitory effect on the enzyme.

Enzyme kinetics in acoustically levitated droplets of supercooled water: a novel approach to cryoenzymology.

PubMed

Weis, David D; Nardozzi, Jonathan D

2005-04-15

The rate of the alkaline phosphatase-catalyzed hydrolysis of 4-methylumbelliferone phosphate was measured in acoustically levitated droplets of aqueous tris (50 mM) at pH 8.5 at 22 +/- 2 degrees C and in supercooled solution at -6 +/- 2 degrees C. At 22 degrees C, the rate of product formation was in excellent agreement with the rate observed in bulk solution in a cuvette, indicating that the acoustic levitation process does not alter the enzyme activity. The rate of the reaction decreased 6-fold in supercooled solution at -6 +/- 2 degrees C. The acoustic levitator apparatus is described in detail.

Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics

PubMed Central

2015-01-01

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings. PMID:26704937

Effects of obesity on liver cytochromes P450 in various animal models.

PubMed

Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

2017-06-01

The prevalence of obesity and other obesity-related diseases is increasing worldwide. Obesity is a disease characterized by increased body weight, or a condition resulting from excessive accumulation of body fat. Due to increased body fat deposits, obesity has also been associated with increased mortality resulting from higher incidence rates of hypertension, diabetes, or various types of cancer, such as breast, colorectal, cervical and prostate cancer. Physiological changes associated with obesity are likely to result in altered drug biotransformation. The main enzymes enabling the oxidative biotransformation of most drugs are cytochromes P450 (CYPs). The review summarizes how pathophysiological factors, especially obesity, affect properties (e.g. enzyme activity, protein expression, gene expression) of CYP enzymes in various experimental models of human obesity. Results reported by various authors suggest that obesity is associated with a decrease of CYP activities (except for the CYP2C and CYP2E1 enzymes). The only exception is mouse obesity induced by monosodium glutamate (administered to newborn mice) as it usually leads to increased CYP expression. Selecting an animal model that is as close as possible to the properties of human obesity is of paramount importance.

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

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

Higgins, M.; Whitworth, G; El Warry, N

2009-01-01

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in themore » other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.« less

Effects of Short-Term Set-Aside Management Practices on Soil Microorganism and Enzyme Activity in China.

PubMed

Li, Guangyu; Wu, Cifang

2017-08-14

Set-aside farmland can effectively improve the self-rehabilitation of arable soil. Long-term set-asides however cannot satisfy provisionment, therefore the use of short-term set-asides to restore cultivated soil is a better option. Few studies have compared short-term set-aside patterns, and the effects of set-asides on soil microbial community and enzyme enzymes. We analyzed the bacterial structure, microbial biomass carbon/nitrogen and enzyme activity of farmland soil under different set-aside regimes in the Yellow River Delta of China. Bacterial alpha diversity was relatively lower under only irrigation, and farmyard manure applications showed clear advantages. Set-asides should consider their influence on soil organic carbon and nitrogen, which were correlated with microbial community structure. Nitrospira (0.47-1.67%), Acidobacteria Gp6 (8.26-15.91%) and unclassified Burkholderiales (1.50-2.81%) were significantly altered ( p < 0.01). Based on functions of these genera, some set-aside patterns led to a relative balance in nitrogen and carbon turnover. Partial treatments showed a deficiency in organic matter. In addition, farmyard manure may lead to the increased consumption of organic matter, with the exception of native plants set-asides. Conventional farming (control group) displayed a significant enzyme activity advantage. Set-aside management practices guided soil microbial communities to different states. Integrated soil microbiota and the content of carbon and nitrogen, native plants with farmyard manure showed an equilibrium state relatively, which would be helpful to improve land quality in the short-term.

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.

PubMed

Sengupta, Shinjinee; Lahiri, Sagar; Banerjee, Shakri; Bashistha, Bipasha; Ghosh, Anil K

2011-12-01

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. 2011 Elsevier B.V. All rights reserved.

Exotic grasses and nitrate enrichment alter soil carbon cycling along an urban-rural tropical forest gradient.

PubMed

Cusack, Daniela F; Lee, Joseph K; McCleery, Taylor L; LeCroy, Chase S

2015-12-01

Urban areas are expanding rapidly in tropical regions, with potential to alter ecosystem dynamics. In particular, exotic grasses and atmospheric nitrogen (N) deposition simultaneously affect tropical urbanized landscapes, with unknown effects on properties like soil carbon (C) storage. We hypothesized that (H1) soil nitrate (NO3 (-) ) is elevated nearer to the urban core, reflecting N deposition gradients. (H2) Exotic grasslands have elevated soil NO3 (-) and decreased soil C relative to secondary forests, with higher N promoting decomposer activity. (H3) Exotic grasslands have greater seasonality in soil NO3 (-) vs. secondary forests, due to higher sensitivity of grassland soil moisture to rainfall. We predicted that NO3 (-) would be positively related to dissolved organic C (DOC) production via changes in decomposer activity. We measured six paired grassland/secondary forest sites along a tropical urban-to-rural gradient during the three dominant seasons (hurricane, dry, and early wet). We found that (1) soil NO3 (-) was generally elevated nearer to the urban core, with particularly clear spatial trends for grasslands. (2) Exotic grasslands had lower soil C than secondary forests, which was related to elevated decomposer enzyme activities and soil respiration. Unexpectedly, soil NO3 (-) was negatively related to enzyme activities, and was lower in grasslands than forests. (3) Grasslands had greater soil NO3 (-) seasonality vs. forests, but this was not strongly linked to shifts in soil moisture or DOC. Our results suggest that exotic grasses in tropical regions are likely to drastically reduce soil C storage, but that N deposition may have an opposite effect via suppression of enzyme activities. However, soil NO3 (-) accumulation here was higher in urban forests than grasslands, potentially related to of aboveground N interception. Net urban effects on C storage across tropical landscapes will likely vary depending on the mosaic of grass cover, rates of N deposition, and responses by local decomposer communities. © 2015 John Wiley & Sons Ltd.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.

PubMed

Sauer, Aisha V; Mrak, Emanuela; Hernandez, Raisa Jofra; Zacchi, Elena; Cavani, Francesco; Casiraghi, Miriam; Grunebaum, Eyal; Roifman, Chaim M; Cervi, Maria C; Ambrosi, Alessandro; Carlucci, Filippo; Roncarolo, Maria Grazia; Villa, Anna; Rubinacci, Alessandro; Aiuti, Alessandro

2009-10-08

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

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

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.

Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue

NASA Astrophysics Data System (ADS)

Yang, Linlin; Li, Lei

2017-03-01

UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that was later found to be > 15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3 4 fold while the G168R mutant by 80 fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2-; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

Differences in the Activities of Eight Enzymes from Ten Soil Fungi and Their Possible Influences on the Surface Structure, Functional Groups, and Element Composition of Soil Colloids

PubMed Central

Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

2014-01-01

How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3–4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11–60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9–22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11–49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance. PMID:25398013

Differences in the activities of eight enzymes from ten soil fungi and their possible influences on the surface structure, functional groups, and element composition of soil colloids.

PubMed

Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

2014-01-01

How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3-4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11-60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9-22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11-49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance.

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

PubMed Central

Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

2015-01-01

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3β enzyme, β-amyloid and tau protein levels.

PubMed

Moyano, Paula; Frejo, María Teresa; Anadon, María José; García, José Manuel; Díaz, María Jesús; Lobo, Margarita; Sola, Emma; García, Jimena; Del Pino, Javier

2018-06-01

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75 NTR and α 7 -nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aβ and tau proteins production, activation of GSK-3β enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3β gene expression, and increases the production of Aβ and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression and purification of spinach nitrite reductase in E. coli

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

Bellissimo, D.; Privalle, L.

1991-03-11

The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth weremore » also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.« less

Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes.

PubMed

Scocca, Sarah; Faustini, Massimo; Villani, Simona; Munari, Eleonora; Conte, Ubaldo; Russo, Vincenzo; Riccardi, Alessia; Vigo, Daniele; Torre, Maria Luisa

2007-04-01

Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil.

PubMed

Mahmoud, Soheil S; Williams, Matthew; Croteau, Rodney

2004-03-01

cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.